U.S. patent application number 15/176106 was filed with the patent office on 2017-01-05 for methods of treating cancer using anti-ox40 antibodies and pd-1 axis binding antagonists.
This patent application is currently assigned to Genentech, Inc.. The applicant listed for this patent is Genentech, Inc.. Invention is credited to Mahrukh HUSENI, Jeong KIM, Chi-Chung LI, Ina P. RHEE, Eric STEFANICH, Sid SUKUMARAN.
Application Number | 20170000885 15/176106 |
Document ID | / |
Family ID | 56134672 |
Filed Date | 2017-01-05 |
United States Patent
Application |
20170000885 |
Kind Code |
A1 |
RHEE; Ina P. ; et
al. |
January 5, 2017 |
METHODS OF TREATING CANCER USING ANTI-OX40 ANTIBODIES AND PD-1 AXIS
BINDING ANTAGONISTS
Abstract
The invention provides methods of treating or delaying
progression of cancer in an individual comprising administering to
the individual an anti-human OX40 agonist antibody and an anti-PDL1
antibody. In some embodiments, the anti-human OX40 agonist antibody
is administered in a dose selected from about 0.8 mg, about 3.2 mg,
about 12 mg, about 40 mg, about 80 mg, about 130 mg, about 160 mg,
about 300 mg, about 320 mg, about 400 mg, about 600 mg, and about
1200 mg, and the anti-PDL1 antibody is administered at a dose of
about 800 mg or about 1200 mg.
Inventors: |
RHEE; Ina P.; (Burlingame,
CA) ; KIM; Jeong; (San Francisco, CA) ;
HUSENI; Mahrukh; (Union City, CA) ; STEFANICH;
Eric; (South San Francisco, CA) ; SUKUMARAN; Sid;
(South San Francisco, CA) ; LI; Chi-Chung; (South
San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genentech, Inc. |
South San Francisco |
CA |
US |
|
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
56134672 |
Appl. No.: |
15/176106 |
Filed: |
June 7, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62336470 |
May 13, 2016 |
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62321679 |
Apr 12, 2016 |
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62308800 |
Mar 15, 2016 |
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62173340 |
Jun 9, 2015 |
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62172803 |
Jun 8, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/545 20130101;
C07K 16/2827 20130101; C07K 16/2878 20130101; C07K 2317/75
20130101; A61K 2039/507 20130101; C07K 2317/24 20130101; A61K
39/3955 20130101; A61K 2039/505 20130101; A61P 35/00 20180101; A61P
43/00 20180101; A61K 9/0019 20130101; C07K 2317/76 20130101 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 9/00 20060101 A61K009/00 |
Claims
1. A method of treating or delaying progression of cancer in an
individual comprising administering to the individual: (i) an
anti-human OX40 agonist antibody at a dose selected from the group
consisting of about 0.8 mg, about 3.2 mg, about 12 mg, about 40 mg,
about 80 mg, about 130 mg, about 160 mg, about 300 mg, about 320
mg, about 400 mg, about 600 mg, and about 1200 mg, wherein the
anti-human OX40 agonist antibody comprises (a) HVR-H1 comprising
the amino acid sequence of SEQ ID NO:2; (b) HVR-H2 comprising the
amino acid sequence of SEQ ID NO:3; (c) HVR-H3 comprising the amino
acid sequence of SEQ ID NO:4; (d) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:6; and (f) HVR-L3 comprising an amino acid
sequence selected from SEQ ID NO:7; and (ii) an anti-PDL1 antibody
at a dose of about 800 mg or about 1200 mg, wherein the anti-PDL1
antibody comprises (a) HVR-H1 comprising the amino acid sequence of
SEQ ID NO:196; (b) HVR-H2 comprising the amino acid sequence of SEQ
ID NO:197; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:198; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:199; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:200; and (f) HVR-L3 comprising an amino acid sequence selected
from SEQ ID NO:201; wherein the individual is a human.
2. The method of claim 1, wherein the anti-human OX40 agonist
antibody is administered at a dose of about 300 mg.
3. The method of claim 1, wherein the anti-human OX40 agonist
antibody and the anti-PDL1 antibody are administered
intravenously.
4. The method of claim 1, wherein the anti-human OX40 agonist
antibody and the anti-PDL1 antibody are administered on the same
day.
5. The method of claim 1, wherein the anti-human OX40 agonist
antibody and the anti-PDL1 antibody are administered on different
days, and wherein the anti-PDL1 antibody is administered within 7
or fewer days of administering the anti-human OX40 agonist
antibody.
6. The method of claim 1, further comprising repeating the
administration of the anti-human OX40 agonist antibody at one or
more additional doses, wherein each dose of the one or more
additional doses is selected from the group consisting of about 0.8
mg, about 3.2 mg, about 12 mg, about 40 mg, about 80 mg, about 130
mg, about 160 mg, about 300 mg, about 320 mg, about 400 mg, about
600 mg, and about 1200 mg per administration and is administered at
an interval of about 2 weeks or about 14 days between each
administration.
7. The method of claim 1, further comprising repeating the
administration of the anti-human OX40 agonist antibody at one or
more additional doses, wherein each dose of the one or more
additional doses is selected from the group consisting of about 0.8
mg, about 3.2 mg, about 12 mg, about 40 mg, about 80 mg, about 130
mg, about 160 mg, about 300 mg, about 320 mg, about 400 mg, about
600 mg, and about 1200 mg per administration and is administered at
an interval of about 3 weeks or about 21 days between each
administration.
8. The method of claim 7, wherein 1-10 additional doses of the
anti-human OX40 agonist antibody are administered.
9. The method of claim 1, further comprising repeating the
administration of the anti-PDL1 antibody at one or more additional
doses, wherein each dose of the one or more additional doses is
about 800 mg and is administered at an interval of about 2 weeks or
about 14 days between each administration.
10. The method of claim 1, further comprising repeating the
administration of the anti-PDL1 antibody at one or more additional
doses, wherein each dose of the one or more additional doses is
about 1200 mg and is administered at an interval of about 3 weeks
or about 21 days between each administration.
11. The method of claim 10, wherein 1-10 additional doses of the
anti-PDL1 antibody are administered.
12. The method of claim 6, wherein each dose of the anti-human OX40
agonist antibody administered to the individual is the same.
13. The method of claim 6, wherein each dose of the anti-human OX40
agonist antibody administered to the individual is not the
same.
14. The method of claim 6, wherein each dose of the anti-human OX40
agonist antibody is administered intravenously.
15. The method of claim 14, wherein a first dose of the anti-human
OX40 agonist antibody is administered to the individual at a first
rate, wherein, after the administration of the first dose, one or
more additional doses of the anti-human OX40 agonist antibody are
administered to the individual at one or more subsequent rates, and
wherein the first rate is slower than the one or more subsequent
rates.
16. The method of claim 10, wherein each dose of the anti-PDL1
antibody is administered intravenously.
17. The method of claim 16, wherein a first dose of the anti-PDL1
antibody is administered to the individual at a first rate,
wherein, after the administration of the first dose, one or more
additional doses of the anti-PDL1 antibody are administered to the
individual at one or more subsequent rates, and wherein the first
rate is slower than the one or more subsequent rates.
18. The method of claim 1, wherein the anti-human OX40 agonist
antibody is a humanized antibody.
19. The method of claim 1, wherein the anti-human OX40 agonist
antibody comprises a heavy chain variable domain (VH) sequence
having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
56, 58, 60, 62, 64, 66, 68, 183, or 184.
20. The method of claim 19, wherein the VH sequence having at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity
contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative to the reference sequence, but an
anti-human OX40 agonist antibody comprising that sequence retains
the ability to bind to human OX40.
21. The method of claim 19, wherein a total of 1 to 10 amino acids
have been substituted, inserted and/or deleted in SEQ ID NO:56.
22. The method of claim 1, wherein the anti-human OX40 agonist
antibody comprises a light chain variable domain (VL) having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 57, 59,
61, 63, 65, 67, or 69.
23. The method of claim 22, wherein the VL sequence having at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity
contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative to the reference sequence, but an
anti-human OX40 agonist antibody comprising that sequence retains
the ability to bind to human OX40.
24. The method of claim 22, wherein a total of 1 to 10 amino acids
have been substituted, inserted and/or deleted in SEQ ID NO:
57.
25. The method of claim 1, wherein the anti-human OX40 agonist
antibody comprises a VH sequence of SEQ ID NO: 56.
26. The method of claim 1, wherein the anti-human OX40 agonist
antibody comprises a VL sequence of SEQ ID NO: 57.
27. The method of claim 1, wherein the anti-human OX40 agonist
antibody comprises a VH sequence of SEQ ID NO:56 and a VL sequence
of SEQ ID NO: 57.
28. The method of claim 1, wherein the anti-human OX40 agonist
antibody is a full length human IgG1 antibody.
29. The method of claim 1, wherein the anti-human OX40 agonist
antibody is MOXR0916.
30. The method of claim 1, wherein the anti-human OX40 agonist
antibody is formulated in a pharmaceutical formulation comprising
(a) the anti-human OX40 agonist antibody at a concentration between
about 10 mg/mL and about 100 mg/mL, (b) a polysorbate, wherein the
polysorbate concentration is about 0.02% to about 0.06%; (c) a
histidine buffer at pH 5.0 to 6.0; and (d) a saccharide, wherein
the saccharide concentration is about 120 mM to about 320 mM.
31. The method of claim 1, wherein the anti-PDL1 antibody is a
monoclonal antibody.
32. The method of claim 1, wherein the anti-PDL1 antibody is an
antibody fragment selected from the group consisting of Fab,
Fab'-SH, Fv, scFv, and (Fab').sub.2 fragments.
33. The method of claim 1, wherein the anti-PDL1 antibody is a
humanized antibody or a human antibody.
34. The method of claim 1, wherein the anti-PDL1 antibody comprises
a human IgG1 having Asn to Ala substitution at position 297
according to EU numbering.
35. The method of claim 1, wherein the anti-PDL1 antibody comprises
a heavy chain variable region comprising the amino acid sequence of
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADS
VKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID
NO:202).
36. The method of claim 1, wherein the anti-PDL1 antibody comprises
a light chain variable region comprising the amino acid sequence of
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIY SASF
LYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID
NO:204).
37. The method of claim 35, wherein the anti-PDL1 antibody
comprises a heavy chain variable region comprising the amino acid
sequence of
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADS
VKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID
NO:202) and a light chain variable region comprising the amino acid
sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIY SASF
LYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID
NO:204).
38. The method of claim 1, wherein the anti-PDL1 antibody comprises
a heavy chain sequence that has at least 85%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% or 100%
sequence identity to the heavy chain sequence: TABLE-US-00021 (SEQ
ID NO: 205) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAW
ISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRH
WPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY
FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI
CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD
TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYAST
YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY
TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.
39. The method of claim 1, wherein the anti-PDL1 antibody comprises
a light chain sequence that has at least 85%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% or 100%
sequence identity to the light chain sequence: TABLE-US-00022 (SEQ
ID NO: 206) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYS
ASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC.
40. The method of claim 1, wherein the anti-PDL1 antibody is
MPDL3280A.
41. The method of claim 1, wherein the method further comprises
administering to the individual an anti-VEGF antibody.
42. The method of claim 41, wherein the anti-VEGF antibody is
bevacizumab.
43. The method of claim 42, wherein bevacizumab is administered to
the individual at a dose of about 15 mg/kg.
44. The method of claim 43, further comprising repeating the
administration of bevacizumab at one or more additional doses,
wherein each dose of the one or more additional doses is about 15
mg/kg and is administered at an interval of about 3 weeks or about
21 days between each administration.
45. The method of claim 41, wherein the anti-human OX40 agonist
antibody, the anti-PDL1 antibody, and anti-VEGF antibody are
administered to the individual on the same day.
46. The method of claim 41, wherein the anti-human OX40 agonist
antibody, the anti-PDL1 antibody, and anti-VEGF antibody are
administered intravenously.
47. The method of claim 1, wherein the treatment results in a
sustained response in the individual after cessation of the
treatment.
48. The method of claim 1, wherein the treatment results in a
complete response (CR) or partial response (PR) in the
individual.
49. The method of claim 1, wherein the individual has a cancer
selected from the group consisting of melanoma, triple-negative
breast cancer, ovarian cancer, renal cell cancer, bladder cancer,
non-small cell lung cancer, gastric cancer, and colorectal
cancer.
50. The method of claim 49, wherein the individual has melanoma,
wherein the melanoma has a BRAF V600 mutation, and wherein, prior
to the administration of the anti-human OX40 agonist antibody and
the anti-PDL1 antibody, the individual has been treated with a
B-Raf and/or mitogen-activated protein kinase kinase (MEK) kinase
inhibitor and exhibited disease progression or intolerance to the
B-Raf and/or mitogen-activated protein kinase kinase (MEK) kinase
inhibitor treatment.
51. The method of claim 49, wherein the individual has non-small
cell lung cancer, wherein the non-small cell lung cancer has a
sensitizing epidermal growth factor receptor (EGFR) mutation, and
wherein, prior to the administration of the anti-human OX40 agonist
antibody and the anti-PDL1 antibody, the individual has been
treated with an EGFR tyrosine kinase inhibitor and exhibited
disease progression or intolerance to the EGFR tyrosine kinase
inhibitor treatment.
52. The method of claim 49, wherein the individual has non-small
cell lung cancer, wherein the non-small cell lung cancer has an
anaplastic lymphoma kinase (ALK) rearrangement, and wherein, prior
to the administration of the anti-human OX40 agonist antibody and
the anti-PDL1 antibody, the individual has been treated with an ALK
tyrosine kinase inhibitor and exhibited disease progression or
intolerance to the ALK tyrosine kinase inhibitor treatment.
53. The method of claim 49, wherein the individual has colorectal
cancer, and wherein the colorectal cancer exhibits microsatellite
instability-high (MSI-H) status.
54. The method of claim 49, wherein the individual has renal cell
cancer, and wherein the renal cell cancer is refractory to a prior
therapy.
55. The method of claim 54, wherein the prior therapy comprises
treatment with a VEGF inhibitor, an mTOR inhibitor, or both.
56. The method of claim 1, wherein the anti-human OX40 agonist
antibody is MOXR0916 administered at a dose of 300 mg, wherein the
anti-PDL1 antibody is atezolizumab administered at a dose of 1200
mg, and wherein the cancer is selected from the group consisting of
melanoma, triple-negative breast cancer, ovarian cancer, renal cell
cancer, bladder cancer, non-small cell lung cancer, gastric cancer,
and colorectal cancer.
57. The method of claim 56, wherein the MOXR0916 and the
atezolizumab are administered on the same day.
58. The method of claim 56, further comprising repeating the
administration of MOXR0916 at a dose of 300 mg per administration,
and repeating the administration of atezolizumab at a dose of 1200
mg per administration, wherein the MOXR0916 and the atezolizumab
are administered at an interval of about 3 weeks or about 21 days
between each administration.
59. The method of claim 58, wherein the repeated administrations of
the MOXR0916 and the atezolizumab are administered on the same
day.
60. The method of claim 56, wherein the MOXR0916 and the
atezolizumab are administered intravenously.
61. The method of claim 1, wherein the method further comprises
administering bevacizumab at a dose of 15 mg/kg, wherein the
anti-human OX40 agonist antibody is MOXR0916 administered at a dose
of 300 mg, wherein the anti-PDL1 antibody is atezolizumab
administered at a dose of 1200 mg, and wherein the cancer is
selected from the group consisting of melanoma, triple-negative
breast cancer, ovarian cancer, renal cell cancer, bladder cancer,
non-small cell lung cancer, gastric cancer, and colorectal
cancer.
62. The method of claim 61, wherein the MOXR0916, the atezolizumab,
and the bevacizumab are administered on the same day.
63. The method of claim 61, further comprising repeating the
administration of MOXR0916 at a dose of 300 mg per administration,
repeating the administration of atezolizumab at a dose of 1200 mg
per administration, and repeating the administration of bevacizumab
at a dose of 15 mg/kg per administration, wherein the MOXR0916, the
atezolizumab, and the bevacizumab are administered at an interval
of about 3 weeks or about 21 days between each administration.
64. The method of claim 63, wherein the repeated administrations of
the MOXR0916, the atezolizumab, and the bevacizumab are
administered on the same day.
65. The method of claim 61, wherein the MOXR0916, the atezolizumab,
and the bevacizumab are administered intravenously.
66. The method of claim 1, further comprising, after administering
to the individual the anti-human OX40 agonist antibody and the
anti-PDL1 antibody, monitoring the responsiveness of the individual
to said treatment by: (a) measuring an expression level of one or
more marker genes in a sample obtained from the cancer of the
individual, wherein the one or more marker genes are selected from
the group consisting of CCR5, CD274, IL-7, TNFRSF14, TGFB1, CD40,
CD4, PRF1, TNFSF4, CD86, CXCL9, CD3E, LAG3, PDCD1, CCL28, GZMB,
IFNg, and IL-2RA; and (b) optionally, classifying the individual as
responsive or non-responsive to treatment with the anti-human OX40
agonist antibody and the anti-PDL1 antibody based on the expression
level of the one or more marker genes in the sample, as compared
with a reference, wherein an increased expression level of the one
or more marker genes as compared with the reference indicates a
responsive individual.
67. The method of claim 1, further comprising, after administering
to the individual the anti-human OX40 agonist antibody and the
anti-PDL1 antibody, monitoring the responsiveness of the individual
to said treatment by: (a) measuring an expression level of one or
more marker genes in a sample obtained from the cancer of the
individual, wherein the one or more marker genes are selected from
the group consisting of CD8b, EOMES, GZMA, GZMB, IFNg, and PRF1;
and (b) optionally, classifying the individual as responsive or
non-responsive to treatment with the anti-human OX40 agonist
antibody and the anti-PDL1 antibody based on the expression level
of the one or more marker genes in the sample, as compared with a
reference, wherein an increased expression level of the one or more
marker genes as compared with the reference indicates a responsive
individual.
68. The method of claim 1, further comprising, after administering
to the individual the anti-human OX40 agonist antibody and the
anti-PDL1 antibody, monitoring the responsiveness of the individual
to said treatment by: (a) measuring an expression level of one or
more marker genes in a sample obtained from the cancer of the
individual, wherein the one or more marker genes are selected from
the group consisting of CCL22, IL-2, RORC, IL-8, CTLA4, and FOXP3;
and (b) optionally, classifying the individual as responsive or
non-responsive to treatment with the anti-human OX40 agonist
antibody and the anti-PDL1 antibody based on the expression level
of the one or more marker genes in the sample, as compared with a
reference, wherein a decreased expression level of the one or more
marker genes as compared with the reference indicates a responsive
individual.
69. The method of claim 1, wherein, prior to the administration of
the anti-human OX40 agonist antibody and the anti-PDL1 antibody,
the individual has been treated with an immunotherapy agent.
70. The method of claim 69, wherein the prior treatment with the
immunotherapy agent is a monotherapy.
71. The method of claim 69, wherein the individual exhibited a
stable disease or disease progression prior to the administration
of the anti-human OX40 agonist antibody and the anti-PDL1
antibody.
72. The method of claim 69, wherein the prior treatment with the
immunotherapy agent comprises treatment with an OX40 agonist in the
absence of a PD-1 axis binding antagonist.
73. The method of claim 72, wherein the OX40 agonist is an
anti-human OX40 agonist antibody.
74. The method of claim 69, wherein the prior treatment with the
immunotherapy agent comprises treatment with a PD-1 axis binding
antagonist in the absence of an OX40 agonist.
75. The method of claim 74, wherein the OX40 agonist is an
anti-human OX40 agonist antibody.
76. The method of claim 72, wherein the PD-1 axis binding
antagonist is an anti-PDL1 antibody.
77. The method of claim 72, wherein the PD-1 axis binding
antagonist is an anti-PD1 antibody.
78. A method for determining whether a cancer patient responds to a
treatment with an anti-human OX40 agonist antibody and an anti-PDL1
antibody, comprising measuring an expression level of one or more
marker genes in a sample obtained from the cancer of the
individual, wherein the one or more marker genes are selected from
the group consisting of CCR5, CD274, IL-7, TNFRSF14, TGFB1, CD40,
CD4, PRF1, TNFSF4, CD86, CXCL9, CD3E, LAG3, PDCD1, CCL28, GZMB,
IFNg, and IL-2RA, wherein the expression level of the one or more
marker genes is compared with a reference, and wherein an increased
expression level of the one or more marker genes as compared with
the reference indicates that the cancer patient responds to said
treatment.
79. A method for determining whether a cancer patient responds to a
treatment with an anti-human OX40 agonist antibody and an anti-PDL1
antibody, comprising measuring an expression level of one or more
marker genes in a sample obtained from the cancer of the
individual, wherein the one or more marker genes are selected from
the group consisting of CD8b, EOMES, GZMA, GZMB, IFNg, and PRF1,
wherein the expression level of the one or more marker genes is
compared with a reference, and wherein an increased expression
level of the one or more marker genes as compared with the
reference indicates that the cancer patient responds to said
treatment.
80. A method for determining whether a cancer patient responds to a
treatment with an anti-human OX40 agonist antibody and an anti-PDL1
antibody, comprising measuring an expression level of one or more
marker genes in a sample obtained from the cancer of the
individual, wherein the one or more marker genes are selected from
the group consisting of CCL22, IL-2, RORC, IL-8, CTLA4, and FOXP3,
wherein the expression level of the one or more marker genes is
compared with a reference, and wherein a decreased expression level
of the one or more marker genes as compared with the reference
indicates that the cancer patient responds to said treatment.
81. A kit for treating or delaying progression of cancer in an
individual, comprising: (i) a container comprising an anti-human
OX40 agonist antibody for administration at a dose selected from
the group consisting of about 0.8 mg, about 3.2 mg, about 12 mg,
about 40 mg, about 80 mg, about 130 mg, about 160 mg, about 300 mg,
about 320 mg, about 400 mg, about 600 mg, and about 1200 mg,
wherein the anti-human OX40 agonist antibody comprises: an HVR-H1
comprising the amino acid sequence of SEQ ID NO:2; an HVR-H2
comprising the amino acid sequence of SEQ ID NO:3; an HVR-H3
comprising the amino acid sequence of SEQ ID NO:4; an HVR-L1
comprising the amino acid sequence of SEQ ID NO:5; an HVR-L2
comprising the amino acid sequence of SEQ ID NO:6; and an HVR-L3
comprising an amino acid sequence selected from SEQ ID NO:7; (ii) a
container comprising an anti-PDL1 antibody for administration at a
dose of about 800 mg or about 1200 mg, wherein the anti-PDL1
antibody comprises (a) HVR-H1 comprising the amino acid sequence of
SEQ ID NO:196; (b) HVR-H2 comprising the amino acid sequence of SEQ
ID NO:197; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:198; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:199; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:200; and (f) HVR-L3 comprising an amino acid sequence selected
from SEQ ID NO:201; and (iii) a package insert with instructions
for treating or delaying progression of cancer in an individual,
wherein the individual is a human.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority benefit of U.S.
Provisional Application Ser. No. 62/172,803, filed Jun. 8, 2015;
62/173,340, filed Jun. 9, 2015; 62/308,800, filed Mar. 15, 2016;
62/321,679, filed Apr. 12, 2016; and 62/336,470, filed May 13,
2016; each of which is incorporated herein by reference in its
entirety.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0002] The content of the following submission on ASCII text file
is incorporated herein by reference in its entirety: a computer
readable form (CRF) of the Sequence Listing (file name:
146392033800SEQLIST.TXT.txt, date recorded: Jun. 1, 2016, size: 167
KB).
FIELD OF THE INVENTION
[0003] The present invention relates to methods of treating cancer
using anti-OX40 antibodies and PD-1 axis binding antagonists (e.g.,
anti-PD-L1 antibodies). In some embodiments, the methods of
treating cancer include administering an anti-OX40 antibody, a PD-1
axis binding antagonist (e.g., anti-PD-L1 antibody), and an
anti-angiogenesis agent (e.g., a VEGF antagonist such as an
anti-VEGF antibody).
BACKGROUND
[0004] The provision of two distinct signals to T-cells is a widely
accepted model for lymphocyte activation of resting T lymphocytes
by antigen-presenting cells (APCs). Lafferty et al, Aust. J. Exp.
Biol. Med. Sci 53: 27-42 (1975). This model further provides for
the discrimination of self from non-self and immune tolerance.
Bretscher et al, Science 169: 1042-1049 (1970); Bretscher, P. A.,
Proc. Nat. Acad. Sci. USA 96: 185-190 (1999); Jenkins et al, J.
Exp. Med. 165: 302-319 (1987). The primary signal, or antigen
specific signal, is transduced through the T-cell receptor (TCR)
following recognition of foreign antigen peptide presented in the
context of the major histocompatibility-complex (MHC). The second
or co-stimulatory signal is delivered to T-cells by co-stimulatory
molecules expressed on antigen-presenting cells (APCs), inducing
T-cells to promote clonal expansion, cytokine secretion and
effector function. Lenschow et al., Ann. Rev. Immunol. 14:233
(1996). In the absence of co-stimulation, T-cells can become
refractory to antigen stimulation, do not mount an effective immune
response, and further may result in exhaustion or tolerance to
foreign antigens.
[0005] In the two-signal model T-cells receive both positive and
negative secondary co-stimulatory signals. The regulation of such
positive and negative signals is critical to maximize the host's
protective immune responses, while maintaining immune tolerance and
preventing autoimmunity. Negative secondary signals seem necessary
for induction of T-cell tolerance, while positive signals promote
T-cell activation. While the simple two-signal model still provides
a valid explanation for naive lymphocytes, a host's immune response
is a dynamic process, and co-stimulatory signals can also be
provided to antigen-exposed T-cells. The mechanism of
co-stimulation is of therapeutic interest because the manipulation
of co-stimulatory signals has shown to provide a means to either
enhance or terminate cell-based immune response. Recently, it has
been discovered that T cell dysfunction or anergy occurs
concurrently with an induced and sustained expression of the
inhibitory receptor, programmed death 1 polypeptide (PD-1). As a
result, therapeutic targeting of PD-1 and other molecules which
signal through interactions with PD-1, such as programmed death
ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2) are an area
of intense interest.
[0006] PD-L1 is overexpressed in many cancers and is often
associated with poor prognosis (Okazaki T et al., Intern. Immun
2007 19(7):813) (Thompson R H et al., Cancer Res 2006, 66(7):3381).
Interestingly, the majority of tumor infiltrating T lymphocytes
predominantly express PD-1, in contrast to T lymphocytes in normal
tissues and peripheral blood T lymphocytes indicating that
up-regulation of PD-1 on tumor-reactive T cells can contribute to
impaired antitumor immune responses (Blood 2009 114(8):1537). This
may be due to exploitation of PD-L1 signaling mediated by PD-L1
expressing tumor cells interacting with PD-1 expressing T cells to
result in attenuation of T cell activation and evasion of immune
surveillance (Sharpe et al., Nat Rev 2002) (Keir M E et al., 2008
Annu. Rev. Immunol. 26:677). Therefore, inhibition of the
PD-L1/PD-1 interaction may enhance CD8+ T cell-mediated killing of
tumors.
[0007] Therapeutic targeting PD-1 and other molecules which signal
through interactions with PD-1, such as programmed death ligand 1
(PD-L1) and programmed death ligand 2 (PD-L2) are an area of
intense interest. The inhibition of PD-L1 signaling has been
proposed as a means to enhance T cell immunity for the treatment of
cancer (e.g., tumor immunity) and infection, including both acute
and chronic (e.g., persistent) infection. An optimal therapeutic
treatment may combine blockade of PD-1 receptor/ligand interaction
with an agent that directly inhibits tumor growth. There remains a
need for an optimal therapy for treating, stabilizing, preventing,
and/or delaying development of various cancers.
[0008] OX40 (also known as CD34, TNFRSF4 and ACT35) is a member of
the tumor necrosis factor receptor superfamily OX40 is not
constitutively expressed on naive T cells, but is induced after
engagement of the T cell receptor (TCR). The ligand for OX40,
OX40L, is predominantly expressed on antigen presenting cells. OX40
is highly expressed by activated CD4+ T cells, activated CD8+ T
cells, memory T cells, and regulatory T cells. OX40 signaling can
provide costimulatory signals to CD4 and CD8 T cells, leading to
enhanced cell proliferation, survival, effector function and
migration. OX40 signaling also enhances memory T cell development
and function.
[0009] Regulatory T cells (Treg) cells are highly enriched in
tumors and tumor draining lymph nodes derived from multiple cancer
indications, including melanoma, NSCLC, renal, ovarian, colon,
pancreatic, hepatocellular, and breast cancer. In a subset of these
indications, increased intratumoral T reg cell densities are
associated with poor patient prognosis, suggesting that these cells
play an important role in suppressing antitumor immunity. OX40
positive tumor infiltrating lymphocytes have been described.
[0010] It is clear that there continues to be a need for agents
that have clinical attributes that are optimal for development as
therapeutic agents. The invention described herein meets these
needs and provides other benefits.
[0011] All references cited herein, including patent applications
and publications, are incorporated by reference in their
entirety.
SUMMARY
[0012] In one aspect, provided herein is a method of treating or
delaying progression of cancer in an individual comprising
administering to the individual: (a) an anti-human OX40 agonist
antibody at a dose selected from the group consisting of about 0.8
mg, about 3.2 mg, about 12 mg, about 40 mg, about 130 mg, about 400
mg, and about 1200 mg, wherein the anti-human OX40 agonist antibody
comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3;
(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; (d)
HVR-L 1 comprising the amino acid sequence of SEQ ID NO:5; (e)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f)
HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:7;
and (b) an anti-PDL1 antibody at a dose of about 1200 mg, wherein
the anti-PDL1 antibody comprises (a) HVR-H1 comprising the amino
acid sequence of SEQ ID NO:196; (b) HVR-H2 comprising the amino
acid sequence of SEQ ID NO:197; (c) HVR-H3 comprising the amino
acid sequence of SEQ ID NO:198; (d) HVR-L1 comprising the amino
acid sequence of SEQ ID NO:199; (e) HVR-L2 comprising the amino
acid sequence of SEQ ID NO:200; and (f) HVR-L3 comprising an amino
acid sequence selected from SEQ ID NO:201; wherein the individual
is a human.
[0013] In another aspect, provided herein is a method of treating
or delaying progression of cancer in an individual comprising
administering to the individual: (i) an anti-human OX40 agonist
antibody at a dose selected from the group consisting of about 0.8
mg, about 3.2 mg, about 12 mg, about 40 mg, about 80 mg, about 130
mg, about 160 mg, about 300 mg, about 320 mg, about 400 mg, about
600 mg, and about 1200 mg, wherein the anti-human OX40 agonist
antibody comprises (a) HVR-H1 comprising the amino acid sequence of
SEQ ID NO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ
ID NO:3; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:4; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5;
(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and
(f) HVR-L3 comprising an amino acid sequence selected from SEQ ID
NO:7; and (ii) an anti-PDL1 antibody at a dose of about 800 mg or
about 1200 mg per administration, wherein the anti-PDL1 antibody
comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:196; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:197; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:198; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:199; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:200; and (f) HVR-L3 comprising an amino acid sequence selected
from SEQ ID NO:201; wherein the individual is a human.
[0014] In some embodiments, the methods further comprise repeating
the administration of the anti-human OX40 agonist antibody at one
or more additional doses, wherein each dose of the one or more
additional doses is selected from the group consisting of about 0.8
mg, about 3.2 mg, about 12 mg, about 40 mg, about 80 mg, about 130
mg, about 160 mg, about 300 mg, about 320 mg, about 400 mg, about
600 mg, and about 1200 mg per administration and is administered at
an interval of about 2 weeks or about 14 days between each
administration. In some embodiments, the methods further comprise
repeating the administration of the anti-human OX40 agonist
antibody at one or more additional doses, wherein each dose of the
one or more additional doses is selected from the group consisting
of about 0.8 mg, about 3.2 mg, about 12 mg, about 40 mg, about 80
mg, about 130 mg, about 160 mg, about 300 mg, about 320 mg, about
400 mg, about 600 mg, and about 1200 mg per administration and is
administered at an interval of about 3 weeks or about 21 days
between each administration.
[0015] In another aspect, provided herein is a kit for treating or
delaying progression of cancer in an individual, comprising: (a) a
container comprising an anti-human OX40 agonist antibody for
administration at a dose selected from the group consisting of 0.8
mg, 3.2 mg, 12 mg, 40 mg, 130 mg, 400 mg, and 1200 mg of the
anti-human OX40 agonist antibody, wherein the anti-human OX40
agonist antibody comprises: an HVR-H1 comprising the amino acid
sequence of SEQ ID NO:2; an HVR-H2 comprising the amino acid
sequence of SEQ ID NO:3; an HVR-H3 comprising the amino acid
sequence of SEQ ID NO:4; an HVR-L1 comprising the amino acid
sequence of SEQ ID NO:5; an HVR-L2 comprising the amino acid
sequence of SEQ ID NO:6; and an HVR-L3 comprising an amino acid
sequence selected from SEQ ID NO:7; (b) a container comprising an
anti-PDL1 antibody for administration at a dose of 1200 mg, wherein
the anti-PDL1 antibody comprises (a) HVR-H1 comprising the amino
acid sequence of SEQ ID NO:196; (b) HVR-H2 comprising the amino
acid sequence of SEQ ID NO:197; (c) HVR-H3 comprising the amino
acid sequence of SEQ ID NO:198; (d) HVR-L1 comprising the amino
acid sequence of SEQ ID NO:199; (e) HVR-L2 comprising the amino
acid sequence of SEQ ID NO:200; and (f) HVR-L3 comprising an amino
acid sequence selected from SEQ ID NO:201; and (c) a package insert
with instructions for treating or delaying progression of cancer in
an individual, wherein the individual is a human.
[0016] In another aspect, provided herein is a kit for treating or
delaying progression of cancer in an individual, comprising: (i) a
container comprising an anti-human OX40 agonist antibody formulated
for administration at a dose selected from the group consisting of
about 0.8 mg, about 3.2 mg, about 12 mg, about 40 mg, about 80 mg,
about 130 mg, about 160 mg, about 300 mg, about 320 mg, about 400
mg, about 600 mg, and about 1200 mg per administration, wherein the
anti-human OX40 agonist antibody comprises: an HVR-H1 comprising
the amino acid sequence of SEQ ID NO:2; an HVR-H2 comprising the
amino acid sequence of SEQ ID NO:3; an HVR-H3 comprising the amino
acid sequence of SEQ ID NO:4; an HVR-L1 comprising the amino acid
sequence of SEQ ID NO:5; an HVR-L2 comprising the amino acid
sequence of SEQ ID NO:6; and an HVR-L3 comprising an amino acid
sequence selected from SEQ ID NO:7; (ii) a container comprising an
anti-PDL1 antibody for administration at a dose of about 800 mg or
about 1200 mg per administration, wherein the anti-PDL1 antibody
comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:196; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:197; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:198; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:199; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:200; and (f) HVR-L3 comprising an amino acid sequence selected
from SEQ ID NO:201; and (iii) a package insert with instructions
for treating or delaying progression of cancer in an individual,
wherein the individual is a human. In some embodiments, the
anti-human OX40 agonist antibody is formulated for administration
at a dose of about 300 mg.
[0017] In some embodiments, any of the kits of the present
disclosure further comprise a container including an anti-VEGF
antibody. In some embodiments, the anti-VEGF antibody is
bevacizumab. In some embodiments, the bevacizumab is formulated for
administration at a dose of about 15 mg/kg. In some embodiments,
any of the kits of the present disclosure further comprise a
package insert with instructions for administering the anti-VEGF
antibody with an anti-human OX40 agonist antibody and an anti-PDL1
antibody.
[0018] In some embodiments, the anti-human OX40 agonist antibody
and the anti-PDL1 antibody are administered intravenously. In some
embodiments, the anti-human OX40 agonist antibody and the anti-PDL1
antibody are administered on the same day. In some embodiments, the
anti-human OX40 agonist antibody and the anti-PDL1 antibody are
administered on different days, and the anti-PDL1 antibody is
administered within 7 or fewer days of administering the anti-human
OX40 agonist antibody. In some embodiments, the anti-human OX40
agonist antibody is administered at a dose of about 300 mg.
[0019] In some embodiments, the methods further comprise (a) after
administering the anti-human OX40 agonist antibody and the
anti-PDL1 antibody, monitoring the individual for an adverse event
and/or efficacy of treatment; and (b) if the individual does not
exhibit an adverse event, and/or if the treatment exhibits
efficacy, administering to the individual: (i) a second dose of the
anti-human OX40 agonist antibody, wherein the second dose of the
anti-human OX40 agonist antibody is selected from the group
consisting of about 0.8 mg, about 3.2 mg, about 12 mg, about 40 mg,
about 130 mg, about 400 mg, and about 1200 mg; and (ii) a second
dose of the anti-PDL1 antibody, wherein the second dose of the
anti-PDL1 antibody is about 1200 mg. In some embodiments, the
methods further comprise administering to the individual: (a) a
second dose of the anti-human OX40 agonist antibody, wherein the
second dose of the anti-human OX40 agonist antibody is selected
from the group consisting of about 0.8 mg, about 3.2 mg, about 12
mg, about 40 mg, about 130 mg, about 400 mg, and about 1200 mg; and
(b) a second dose of the anti-PDL1 antibody, wherein the second
dose of the anti-PDL1 antibody is about 1200 mg; wherein the second
dose of the anti-human OX40 agonist antibody is not provided until
from about 2 weeks to about 4 weeks after the first dose of the
anti-human OX40 agonist antibody; and wherein the second dose of
the anti-PDL1 antibody is not provided until from about 2 weeks to
about 4 weeks after the first dose of the anti-PDL1 antibody. In
some embodiments, the first dose of the anti-human OX40 agonist
antibody and the first dose of the anti-PDL1 antibody are
administered on the same day, the second dose of the anti-human
OX40 agonist antibody and the second dose of the anti-PDL1 antibody
are administered on the same day, and the second dose of the
anti-human OX40 agonist antibody and the second dose of the
anti-PDL1 antibody are not provided until about 3 weeks after the
first dose of the anti-human OX40 agonist antibody and the first
dose of the anti-human OX40 agonist antibody. In some embodiments,
the first dose of the anti-human OX40 agonist antibody and the
first dose of the anti-PDL1 antibody are administered on the same
day, the second dose of the anti-human OX40 agonist antibody and
the second dose of the anti-PDL1 antibody are administered on the
same day, and the second dose of the anti-human OX40 agonist
antibody and the second dose of the anti-PDL1 antibody are not
provided until about 21 days after the first dose of the anti-human
OX40 agonist antibody and the first dose of the anti-human OX40
agonist antibody. In some embodiments, the second dose of the
anti-human OX40 agonist antibody is greater than the first dose of
the anti-human OX40 agonist antibody. In some embodiments, the
first dose of the anti-human OX40 agonist antibody, the first dose
of the anti-PDL1 antibody, the second dose of the anti-human OX40
agonist antibody, and the second dose of the anti-PDL1 antibody are
administered intravenously.
[0020] In another aspect, provided herein is a method of treating
or delaying progression of cancer in an individual comprising
administering to the individual: (i) an anti-human OX40 agonist
antibody at a dose selected from the group consisting of about 0.8
mg, about 3.2 mg, about 12 mg, about 40 mg, about 80 mg, about 130
mg, about 160 mg, about 300 mg, about 320 mg, about 400 mg, about
600 mg, and about 1200 mg, wherein the anti-human OX40 agonist
antibody comprises (a) HVR-H1 comprising the amino acid sequence of
SEQ ID NO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ
ID NO:3; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:4; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5;
(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and
(f) HVR-L3 comprising an amino acid sequence selected from SEQ ID
NO:7; and (ii) an anti-PDL1 antibody at a dose of about 1200 mg,
wherein the anti-PDL1 antibody comprises (a) HVR-H1 comprising the
amino acid sequence of SEQ ID NO:196; (b) HVR-H2 comprising the
amino acid sequence of SEQ ID NO:197; (c) HVR-H3 comprising the
amino acid sequence of SEQ ID NO:198; (d) HVR-L1 comprising the
amino acid sequence of SEQ ID NO:199; (e) HVR-L2 comprising the
amino acid sequence of SEQ ID NO:200; and (f) HVR-L3 comprising an
amino acid sequence selected from SEQ ID NO:201; wherein the
individual is a human. In some embodiments, the methods further
comprise repeating the administration of the anti-human OX40
agonist antibody and/or the anti-PDL1 antibody at an interval of
about 3 weeks or about 21 days between each administration. In some
embodiments, the anti-human OX40 agonist antibody is administered
at a dose of about 300 mg.
[0021] In another aspect, provided herein is a kit for treating or
delaying progression of cancer in an individual, comprising: (i) a
container comprising an anti-human OX40 agonist antibody formulated
for administration at an interval of about 3 weeks or about 21 days
between each administration at a dose selected from the group
consisting of about 0.8 mg, about 3.2 mg, about 12 mg, about 40 mg,
about 80 mg, about 130 mg, about 160 mg, about 300 mg, about 320
mg, about 400 mg, about 600 mg, and about 1200 mg per
administration, wherein the anti-human OX40 agonist antibody
comprises: an HVR-H1 comprising the amino acid sequence of SEQ ID
NO:2; an HVR-H2 comprising the amino acid sequence of SEQ ID NO:3;
an HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; an
HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; an HVR-L2
comprising the amino acid sequence of SEQ ID NO:6; and an HVR-L3
comprising an amino acid sequence selected from SEQ ID NO:7; (ii) a
container comprising an anti-PDL1 antibody formulated for
administration at an interval of about 3 weeks or about 21 days
between each administration at a dose of about 1200 mg per
administration, wherein the anti-PDL1 antibody comprises (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO:196; (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO:197; (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:198; (d) HVR-L1
comprising the amino acid sequence of SEQ ID NO:199; (e) HVR-L2
comprising the amino acid sequence of SEQ ID NO:200; and (f) HVR-L3
comprising an amino acid sequence selected from SEQ ID NO:201; and
(iii) a package insert with instructions for treating or delaying
progression of cancer in an individual, wherein the individual is a
human.
[0022] In another aspect, provided herein is a method of treating
or delaying progression of cancer in an individual comprising
administering to the individual: (i) an anti-human OX40 agonist
antibody at a dose selected from the group consisting of about 0.5
mg, about 2 mg, about 8 mg, about 27 mg, about 53 mg, about 87 mg,
about 107 mg, about 200 mg, about 213 mg, about 267 mg, about 400
mg, and about 800 mg, wherein the anti-human OX40 agonist antibody
comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3;
(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; (d)
HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f)
HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:7;
and (ii) an anti-PDL1 antibody at a dose of about 800 mg, wherein
the anti-PDL1 antibody comprises (a) HVR-H1 comprising the amino
acid sequence of SEQ ID NO:196; (b) HVR-H2 comprising the amino
acid sequence of SEQ ID NO:197; (c) HVR-H3 comprising the amino
acid sequence of SEQ ID NO:198; (d) HVR-L1 comprising the amino
acid sequence of SEQ ID NO:199; (e) HVR-L2 comprising the amino
acid sequence of SEQ ID NO:200; and (f) HVR-L3 comprising an amino
acid sequence selected from SEQ ID NO:201; wherein the individual
is a human. In some embodiments, the methods further comprise
repeating the administration of the anti-human OX40 agonist
antibody and/or the anti-PDL1 antibody at an interval of about 2
weeks or about 14 days between each administration.
[0023] In another aspect, provided herein is a kit for treating or
delaying progression of cancer in an individual, comprising: (i) a
container comprising an anti-human OX40 agonist antibody formulated
for administration at an interval of about 2 weeks or about 14 days
between each administration at a dose selected from the group
consisting of about 0.5 mg, about 2 mg, about 8 mg, about 27 mg,
about 53 mg, about 87 mg, about 107 mg, about 200 mg, about 213 mg,
about 267 mg, about 400 mg, and about 800 mg per administration,
wherein the anti-human OX40 agonist antibody comprises: an HVR-H1
comprising the amino acid sequence of SEQ ID NO:2; an HVR-H2
comprising the amino acid sequence of SEQ ID NO:3; an HVR-H3
comprising the amino acid sequence of SEQ ID NO:4; an HVR-L1
comprising the amino acid sequence of SEQ ID NO:5; an HVR-L2
comprising the amino acid sequence of SEQ ID NO:6; and an HVR-L3
comprising an amino acid sequence selected from SEQ ID NO:7; (ii) a
container comprising an anti-PDL1 antibody formulated for
administration at an interval of about 2 weeks or about 14 days
between each administration at a dose of about 800 mg per
administration, wherein the anti-PDL1 antibody comprises (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO:196; (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO:197; (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:198; (d) HVR-L1
comprising the amino acid sequence of SEQ ID NO:199; (e) HVR-L2
comprising the amino acid sequence of SEQ ID NO:200; and (f) HVR-L3
comprising an amino acid sequence selected from SEQ ID NO:201; and
(iii) a package insert with instructions for treating or delaying
progression of cancer in an individual, wherein the individual is a
human.
[0024] In some embodiments, 1-10 additional doses of the anti-human
OX40 agonist antibody are administered.
[0025] In some embodiments, the methods further comprise repeating
the administration of the anti-PDL1 antibody at one or more
additional doses, wherein each dose of the one or more additional
doses is about 800 mg and is administered at an interval of about 2
weeks or about 14 days between each administration. In some
embodiments, the methods further comprise repeating the
administration of the anti-PDL1 antibody at one or more additional
doses, wherein each dose of the one or more additional doses is
about 1200 mg and is administered at an interval of about 3 weeks
or about 21 days between each administration. In some embodiments,
1-10 additional doses of the anti-PDL1 antibody are
administered.
[0026] In some embodiments, each dose of the anti-human OX40
agonist antibody administered to the individual is the same. In
some embodiments, each dose of the anti-human OX40 agonist antibody
administered to the individual is not the same. In some
embodiments, each dose of the anti-human OX40 agonist antibody is
administered intravenously. In some embodiments, a first dose of
the anti-human OX40 agonist antibody is administered to the
individual at a first rate, wherein, after the administration of
the first dose, one or more additional doses of the anti-human OX40
agonist antibody are administered to the individual at one or more
subsequent rates, and wherein the first rate is slower than the one
or more subsequent rates. In some embodiments, each dose of the
anti-PDL1 antibody is administered intravenously. In some
embodiments, a first dose of the anti-PDL1 antibody is administered
to the individual at a first rate, wherein, after the
administration of the first dose, one or more additional doses of
the anti-PDL1 antibody are administered to the individual at one or
more subsequent rates, and wherein the first rate is slower than
the one or more subsequent rates.
[0027] In some embodiments, the anti-human OX40 agonist antibody is
a human or humanized antibody. In some embodiments, the anti-human
OX40 agonist antibody comprises a heavy chain variable domain (VH)
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 183, or 184. In some
embodiments, the VH sequence having at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions
(e.g., conservative substitutions), insertions, or deletions
relative to the reference sequence, but an anti-human OX40 agonist
antibody comprising that sequence retains the ability to bind to
human OX40. In some embodiments, the anti-human OX40 agonist
antibody comprises a light chain variable domain (VL) having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 57, 59,
61, 63, 65, 67, or 69. In some embodiments, the VL sequence having
at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative to the reference sequence, but an
anti-human OX40 agonist antibody comprising that sequence retains
the ability to bind to human OX40. In some embodiments, a total of
1 to 10 amino acids have been substituted, inserted and/or deleted
in SEQ ID NO: 57. In some embodiments, the anti-human OX40 agonist
antibody comprises a VH sequence of SEQ ID NO: 56. In some
embodiments, the anti-human OX40 agonist antibody comprises a VL
sequence of SEQ ID NO: 57. In some embodiments, the anti-human OX40
agonist antibody comprises a VH sequence of SEQ ID NO:56 and a VL
sequence of SEQ ID NO: 57. In some embodiments, the anti-human OX40
agonist antibody is a full length human IgG1 antibody. In some
embodiments, the anti-human OX40 agonist antibody is MOXR0916.
[0028] In some embodiments, the anti-human OX40 agonist antibody is
formulated in a pharmaceutical formulation comprising (a) the
anti-human OX40 agonist antibody at a concentration between about
10 mg/mL and about 100 mg/mL, (b) a polysorbate, wherein the
polysorbate concentration is about 0.02% to about 0.06%; (c) a
histidine buffer at pH 5.0 to 6.0; and (d) a saccharide, wherein
the saccharide concentration is about 120 mM to about 320 mM.
[0029] In some embodiments, the anti-PDL1 antibody is a monoclonal
antibody. In some embodiments, the anti-PDL1 antibody is an
antibody fragment selected from the group consisting of Fab,
Fab'-SH, Fv, scFv, and (Fab').sub.2 fragments. In some embodiments,
the anti-PDL1 antibody is a humanized antibody or a human antibody.
In some embodiments, the anti-PDL1 antibody comprises a human IgG1
having Asn to Ala substitution at position 297 according to EU
numbering. In some embodiments, the anti-PDL1 antibody comprises a
heavy chain variable region comprising the amino acid sequence of
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADS
VKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID
NO:202) or EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWI
SPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTL
VTVSSASTK (SEQ ID NO:203). In some embodiments, the anti-PDLL
antibody comprises a light chain variable region comprising the
amino acid sequence of
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIY SASF
LYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID
NO:204). In some embodiments, the anti-PDL1 antibody comprises a
heavy chain variable region comprising the amino acid sequence of
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADS
VKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID
NO:202) or EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWI
SPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTL
VTVSSASTK (SEQ ID NO:203) and a light chain variable region
comprising the amino acid sequence of
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIY SASF
LYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID
NO:204). In some embodiments, the anti-PDL1 antibody comprises a
heavy chain sequence that has at least 85%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99% or 100%
sequence identity to the heavy chain sequence:
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADS
VKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSV
FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS
SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP
EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ
ID NO:205). In some embodiments, the anti-PDLL antibody comprises a
light chain sequence that has at least 85%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99% or 100%
sequence identity to the light chain sequence:
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGS
GSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC
EVTHQGLSSPVTKSFNRGEC (SEQ ID NO:206). In some embodiments, the
anti-PDL1 antibody is MPDL3280A.
[0030] In some embodiments, any of the methods described herein
further comprise administering to the individual an
anti-angiogenesis agent. In some embodiments, any of the methods
described herein further comprise administering to the individual
an anti-VEGF antibody. In some embodiments, the anti-VEGF antibody
is bevacizumab. In some embodiments, bevacizumab is administered to
the individual at a dose of about 15 mg/kg. In some embodiments,
any of the methods described herein further comprise repeating the
administration of bevacizumab at one or more additional doses,
wherein each dose of the one or more additional doses is about 15
mg/kg and is administered at an interval of about 3 weeks or about
21 days between each administration. In some embodiments, any of
the methods described herein further comprise administering the
anti-human OX40 agonist antibody, the anti-PDL1 antibody, and the
anti-VEGF antibody to the individual by intravenous infusion on the
same day.
[0031] In some embodiments, the treatment results in a sustained
response in the individual after cessation of the treatment. In
some embodiments, the treatment results in a complete response (CR)
or partial response (PR) in the individual.
[0032] In some embodiments, the individual has a cancer selected
from the group consisting of melanoma, triple-negative breast
cancer, ovarian cancer, renal cell cancer, bladder cancer,
non-small cell lung cancer, gastric cancer, and colorectal cancer.
In some embodiments, the individual has melanoma, the melanoma has
a BRAF V600 mutation, and, prior to the administration of the
anti-human OX40 agonist antibody, the individual has been treated
with a B-Raf and/or mitogen-activated protein kinase kinase (MEK)
kinase inhibitor and exhibited disease progression or intolerance
to the B-Raf and/or mitogen-activated protein kinase kinase (MEK)
kinase inhibitor treatment. In some embodiments, the individual has
non-small cell lung cancer, the non-small cell lung cancer has a
sensitizing epidermal growth factor receptor (EGFR) mutation, and,
prior to the administration of the anti-human OX40 agonist
antibody, the individual has been treated with an EGFR tyrosine
kinase inhibitor and exhibited disease progression or intolerance
to the EGFR tyrosine kinase inhibitor treatment. In some
embodiments, the individual has non-small cell lung cancer, the
non-small cell lung cancer has an anaplastic lymphoma kinase (ALK)
rearrangement, and, prior to the administration of the anti-human
OX40 agonist antibody, the individual has been treated with an ALK
tyrosine kinase inhibitor and exhibited disease progression or
intolerance to the ALK tyrosine kinase inhibitor treatment. In some
embodiments, the individual has colorectal cancer, and the
colorectal cancer exhibits microsatellite instability-high (MSI-H)
status. In some embodiments, the individual has renal cell cancer,
and the renal cell cancer is refractory to a prior therapy. In some
embodiments, the prior therapy comprises treatment with a VEGF
inhibitor, an mTOR inhibitor, or both.
[0033] In some embodiments, prior to the administration of the
anti-human OX40 agonist antibody and the anti-PDL1 antibody, the
individual has been previously treated with an immunotherapy agent.
In some embodiments, the prior treatment with the immunotherapy
agent is a monotherapy. In some embodiments, the individual
exhibited a stable disease or disease progression prior to the
administration of the anti-human OX40 agonist antibody and the
anti-PDL1 antibody. In some embodiments, the prior treatment with
the immunotherapy agent comprises treatment with an OX40 agonist in
the absence of a PD-1 axis binding antagonist. In some embodiments,
the OX40 agonist is an anti-human OX40 agonist antibody. In some
embodiments, the prior treatment with the immunotherapy agent
comprises treatment with a PD-1 axis binding antagonist in the
absence of an OX40 agonist. In some embodiments, the OX40 agonist
is an anti-human OX40 agonist antibody. In some embodiments, the
PD-1 axis binding antagonist is an anti-PDL1 antibody. In some
embodiments, the PD-1 axis binding antagonist is an anti-PD1
antibody.
[0034] In another aspect, provided herein is a use of an anti-human
OX40 agonist antibody in the manufacture of a first medicament for
treating or delaying progression of cancer in an individual in
conjunction with a second medicament, wherein the first medicament
comprises an anti-human OX40 agonist antibody formulated at a dose
selected from the group consisting of about 0.8 mg, about 3.2 mg,
about 12 mg, about 40 mg, about 80 mg, about 130 mg, about 160 mg,
about 300 mg, about 320 mg, about 400 mg, about 600 mg, and about
1200 mg per administration, wherein the anti-human OX40 agonist
antibody comprises (a) HVR-H1 comprising the amino acid sequence of
SEQ ID NO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ
ID NO:3; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:4; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5;
(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and
(f) HVR-L3 comprising an amino acid sequence selected from SEQ ID
NO:7; and wherein the second medicament comprises an anti-PDL1
antibody formulated at a dose of about 800 mg or about 1200 mg per
administration, wherein the anti-PDL1 antibody comprises (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO:196; (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO:197; (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:198; (d) HVR-L1
comprising the amino acid sequence of SEQ ID NO:199; (e) HVR-L2
comprising the amino acid sequence of SEQ ID NO:200; and (f) HVR-L3
comprising an amino acid sequence selected from SEQ ID NO:201. In
some embodiments, the individual is a human. In some embodiments,
anti-human OX40 agonist antibody is formulated for administration
at a dose of about 300 mg.
[0035] In another aspect, provided herein is a use of an anti-PDL1
antibody in the manufacture of a first medicament for treating or
delaying progression of cancer in an individual in conjunction with
a second medicament, wherein the first medicament comprises an
anti-PDL1 antibody formulated at a dose of about 800 mg or about
1200 mg per administration, wherein the anti-PDL1 antibody
comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:196; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:197; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:198; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:199; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:200; and (f) HVR-L3 comprising an amino acid sequence selected
from SEQ ID NO:201; and wherein the second medicament comprises an
anti-human OX40 agonist antibody formulated at a dose selected from
the group consisting of about 0.8 mg, about 3.2 mg, about 12 mg,
about 40 mg, about 80 mg, about 130 mg, about 160 mg, about 300 mg,
about 320 mg, about 400 mg, about 600 mg, and about 1200 mg per
administration, wherein the anti-human OX40 agonist antibody
comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3;
(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; (d)
HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f)
HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:7.
In some embodiments, the individual is a human. In some
embodiments, anti-human OX40 agonist antibody is formulated for
administration at a dose of about 300 mg.
[0036] In another aspect, provided herein is a use of an anti-VEGF
antibody in the manufacture of a first medicament for treating or
delaying progression of cancer in an individual in conjunction with
a second and a third medicament, wherein the first medicament
comprises bevacizumab formulated at a dose of about 15 mg/kg,
wherein the second medicament comprises an anti-PDL1 antibody
formulated at a dose of about 800 mg or about 1200 mg per
administration, wherein the anti-PDL1 antibody comprises (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO:196; (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO:197; (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:198; (d) HVR-L1
comprising the amino acid sequence of SEQ ID NO:199; (e) HVR-L2
comprising the amino acid sequence of SEQ ID NO:200; and (f) HVR-L3
comprising an amino acid sequence selected from SEQ ID NO:201; and
wherein the third medicament comprises an anti-human OX40 agonist
antibody formulated at a dose selected from the group consisting of
about 0.8 mg, about 3.2 mg, about 12 mg, about 40 mg, about 80 mg,
about 130 mg, about 160 mg, about 300 mg, about 320 mg, about 400
mg, about 600 mg, and about 1200 mg per administration, wherein the
anti-human OX40 agonist antibody comprises (a) HVR-H1 comprising
the amino acid sequence of SEQ ID NO:2; (b) HVR-H2 comprising the
amino acid sequence of SEQ ID NO:3; (c) HVR-H3 comprising the amino
acid sequence of SEQ ID NO:4; (d) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:6; and (f) HVR-L3 comprising an amino acid
sequence selected from SEQ ID NO:7.
[0037] In another aspect, provided herein is a use of an anti-human
OX40 agonist antibody in the manufacture of a first medicament for
treating or delaying progression of cancer in an individual in
conjunction with a second medicament, wherein the first medicament
comprises an anti-human OX40 agonist antibody formulated for
administration at an interval of about 3 weeks or about 21 days
between each administration at a dose selected from the group
consisting of about 0.8 mg, about 3.2 mg, about 12 mg, about 40 mg,
about 80 mg, about 130 mg, about 160 mg, about 300 mg, about 320
mg, about 400 mg, about 600 mg, and about 1200 mg per
administration, wherein the anti-human OX40 agonist antibody
comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3;
(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; (d)
HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f)
HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:7;
and wherein the second medicament comprises an anti-PDL1 antibody
formulated for administration at an interval of about 3 weeks or
about 21 days between each administration at a dose of about 1200
mg per administration, wherein the anti-PDL1 antibody comprises (a)
HVR-H1 comprising the amino acid sequence of SEQ ID NO:196; (b)
HVR-H2 comprising the amino acid sequence of SEQ ID NO:197; (c)
HVR-H3 comprising the amino acid sequence of SEQ ID NO:198; (d)
HVR-L1 comprising the amino acid sequence of SEQ ID NO:199; (e)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:200; and (f)
HVR-L3 comprising an amino acid sequence selected from SEQ ID
NO:201. In some embodiments, the individual is a human. In some
embodiments, anti-human OX40 agonist antibody is formulated for
administration at a dose of about 300 mg.
[0038] In another aspect, provided herein is a use of an anti-PDL1
antibody in the manufacture of a first medicament for treating or
delaying progression of cancer in an individual in conjunction with
a second medicament, wherein the first medicament comprises an
anti-PDL1 antibody formulated for administration at an interval of
about 3 weeks or about 21 days between each administration at a
dose of about 1200 mg per administration, wherein the anti-PDL1
antibody comprises (a) HVR-H1 comprising the amino acid sequence of
SEQ ID NO:196; (b) HVR-H2 comprising the amino acid sequence of SEQ
ID NO:197; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:198; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:199; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:200; and (f) HVR-L3 comprising an amino acid sequence selected
from SEQ ID NO:201; and wherein the second medicament comprises an
anti-human OX40 agonist antibody formulated for administration at
an interval of about 3 weeks or about 21 days between each
administration at a dose selected from the group consisting of
about 0.8 mg, about 3.2 mg, about 12 mg, about 40 mg, about 80 mg,
about 130 mg, about 160 mg, about 300 mg, about 320 mg, about 400
mg, about 600 mg, and about 1200 mg per administration, wherein the
anti-human OX40 agonist antibody comprises (a) HVR-H1 comprising
the amino acid sequence of SEQ ID NO:2; (b) HVR-H2 comprising the
amino acid sequence of SEQ ID NO:3; (c) HVR-H3 comprising the amino
acid sequence of SEQ ID NO:4; (d) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:6; and (f) HVR-L3 comprising an amino acid
sequence selected from SEQ ID NO:7. In some embodiments, the
individual is a human. In some embodiments, anti-human OX40 agonist
antibody is formulated for administration at a dose of about 300
mg.
[0039] In another aspect, provided herein is a use of an anti-human
OX40 agonist antibody in the manufacture of a first medicament for
treating or delaying progression of cancer in an individual in
conjunction with a second medicament, wherein the first medicament
comprises an anti-human OX40 agonist antibody formulated for
administration at an interval of about 2 weeks or about 14 days
between each administration at a dose selected from the group
consisting of about 0.5 mg, about 2 mg, about 8 mg, about 27 mg,
about 53 mg, about 87 mg, about 107 mg, about 200 mg, about 213 mg,
about 267 mg, about 400 mg, and about 800 mg per administration,
wherein the anti-human OX40 agonist antibody comprises (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO:2; (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO:3; (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:4; (d) HVR-L1
comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2
comprising the amino acid sequence of SEQ ID NO:6; and (f) HVR-L3
comprising an amino acid sequence selected from SEQ ID NO:7; and
wherein the second medicament comprises an anti-PDL1 antibody
formulated for administration at an interval of about 2 weeks or
about 14 days between each administration at a dose of about 800 mg
per administration, wherein the anti-PDL1 antibody comprises (a)
HVR-H1 comprising the amino acid sequence of SEQ ID NO:196; (b)
HVR-H2 comprising the amino acid sequence of SEQ ID NO:197; (c)
HVR-H3 comprising the amino acid sequence of SEQ ID NO:198; (d)
HVR-L1 comprising the amino acid sequence of SEQ ID NO:199; (e)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:200; and (f)
HVR-L3 comprising an amino acid sequence selected from SEQ ID
NO:201. In some embodiments, the individual is a human. In some
embodiments, anti-human OX40 agonist antibody is formulated for
administration at a dose of about 300 mg.
[0040] In another aspect, provided herein is a use of an anti-PDL1
antibody in the manufacture of a first medicament for treating or
delaying progression of cancer in an individual in conjunction with
a second medicament, wherein the first medicament comprises an
anti-PDL1 antibody formulated for administration at an interval of
about 2 weeks or about 14 days between each administration at a
dose of about 800 mg per administration, wherein the anti-PDL1
antibody comprises (a) HVR-H1 comprising the amino acid sequence of
SEQ ID NO:196; (b) HVR-H2 comprising the amino acid sequence of SEQ
ID NO:197; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:198; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:199; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:200; and (f) HVR-L3 comprising an amino acid sequence selected
from SEQ ID NO:201; and wherein the second medicament comprises an
anti-human OX40 agonist antibody formulated for administration at
an interval of about 2 weeks or about 14 days between each
administration at a dose selected from the group consisting of
about 0.5 mg, about 2 mg, about 8 mg, about 27 mg, about 53 mg,
about 87 mg, about 107 mg, about 200 mg, about 213 mg, about 267
mg, about 400 mg, and about 800 mg per administration, wherein the
anti-human OX40 agonist antibody comprises (a) HVR-H1 comprising
the amino acid sequence of SEQ ID NO:2; (b) HVR-H2 comprising the
amino acid sequence of SEQ ID NO:3; (c) HVR-H3 comprising the amino
acid sequence of SEQ ID NO:4; (d) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:6; and (f) HVR-L3 comprising an amino acid
sequence selected from SEQ ID NO:7. In some embodiments, the
individual is a human. In some embodiments, anti-human OX40 agonist
antibody is formulated for administration at a dose of about 300
mg.
[0041] In some embodiments, provided herein is a method of treating
or delaying progression of cancer in an individual comprising
administering to the individual (i) MOXR0916 at a dose of about 300
mg, and (ii) atezolizumab at a dose of about 1200 mg, wherein the
cancer is selected from the group consisting of melanoma,
triple-negative breast cancer, ovarian cancer, renal cell cancer,
bladder cancer, non-small cell lung cancer, gastric cancer, and
colorectal cancer. In some embodiments, the MOXR0916 and
atezolizumab are administered on the same day. In some embodiments,
the method further comprises repeating the administration of
MOXR0916 at a dose of about 300 mg per administration and
atezolizumab at a dose of about 1200 mg per administration, and
wherein the administration is repeated at an interval of about 3
weeks or about 21 days between administrations. In some
embodiments, the repeated administrations of the MOXR0916 and the
atezolizumab are administered on the same day. In some embodiments,
the cancer is RCC. In some embodiments, the cancer is bladder
cancer. In some embodiments, MOXR0916 is administered
intravenously. In some embodiments, atezolizumab is administered
intravenously. In some embodiments, MOXR0916 and atezolizumab are
administered intravenously.
[0042] In some embodiments, provided herein is a method of treating
or delaying progression of cancer in an individual comprising
administering to the individual (i) MOXR0916 at a dose of about 160
mg, and (ii) atezolizumab at a dose of about 1200 mg, wherein the
cancer is selected from the group consisting of melanoma,
triple-negative breast cancer, ovarian cancer, renal cell cancer,
bladder cancer, non-small cell lung cancer, gastric cancer, and
colorectal cancer. In some embodiments, the MOXR0916 and
atezolizumab are administered on the same day. In some embodiments,
the method further comprises repeating the administration of
MOXR0916 at a dose of about 160 mg per administration and
atezolizumab at a dose of about 1200 mg per administration, and
wherein the administration is repeated at an interval of about 3
weeks or about 21 days between administrations. In some
embodiments, the repeated administrations of the MOXR0916 and the
atezolizumab are administered on the same day. In some embodiments,
the cancer is RCC. In some embodiments, the cancer is bladder
cancer. In some embodiments, MOXR0916 is administered
intravenously. In some embodiments, atezolizumab is administered
intravenously. In some embodiments, MOXR0916 and atezolizumab are
administered intravenously.
[0043] In some embodiments, provided herein is a method of treating
or delaying progression of cancer in an individual comprising
administering to the individual (i) MOXR0916 at a dose of about 320
mg, and (ii) atezolizumab at a dose of about 1200 mg, wherein the
cancer is selected from the group consisting of melanoma,
triple-negative breast cancer, ovarian cancer, renal cell cancer,
bladder cancer, non-small cell lung cancer, gastric cancer, and
colorectal cancer. In some embodiments, the MOXR0916 and
atezolizumab are administered on the same day. In some embodiments,
the method further comprises repeating the administration of
MOXR0916 at a dose of about 320 mg per administration and
atezolizumab at a dose of about 1200 mg per administration, and
wherein the administration is repeated at an interval of about 3
weeks or about 21 days between administrations. In some
embodiments, the repeated administrations of the MOXR0916 and the
atezolizumab are administered on the same day. In some embodiments,
the cancer is RCC. In some embodiments, the cancer is bladder
cancer. In some embodiments, MOXR0916 is administered
intravenously. In some embodiments, atezolizumab is administered
intravenously. In some embodiments, MOXR0916 and atezolizumab are
administered intravenously.
[0044] In some embodiments, provided herein is a method of treating
or delaying progression of cancer in an individual comprising
administering to the individual (i) MOXR0916 at a dose of about 400
mg, and (ii) atezolizumab at a dose of about 1200 mg, wherein the
cancer is selected from the group consisting of melanoma,
triple-negative breast cancer, ovarian cancer, renal cell cancer,
bladder cancer, non-small cell lung cancer, gastric cancer, and
colorectal cancer. In some embodiments, the MOXR0916 and
atezolizumab are administered on the same day. In some embodiments,
the method further comprises repeating the administration of
MOXR0916 at a dose of about 400 mg per administration and
atezolizumab at a dose of about 1200 mg per administration, and
wherein the administration is repeated at an interval of about 3
weeks or about 21 days between administrations. In some
embodiments, the repeated administrations of the MOXR0916 and the
atezolizumab are administered on the same day. In some embodiments,
the cancer is RCC. In some embodiments, the cancer is bladder
cancer. In some embodiments, MOXR0916 is administered
intravenously. In some embodiments, atezolizumab is administered
intravenously. In some embodiments, MOXR0916 and atezolizumab are
administered intravenously.
[0045] In some embodiments, provided herein is a method of treating
or delaying progression of cancer in an individual comprising
administering to the individual (i) MOXR0916 at a dose of about 300
mg, (ii) atezolizumab at a dose of about 1200 mg, and (iii)
bevacizumab at a dose of about 15 mg/kg, wherein the cancer is
selected from the group consisting of melanoma, triple-negative
breast cancer, ovarian cancer, renal cell cancer, bladder cancer,
non-small cell lung cancer, gastric cancer, and colorectal cancer.
In some embodiments, the MOXR0916, atezolizumab, and bevacizumab
are administered on the same day. In some embodiments, the method
further comprises repeating the administration of MOXR0916 at a
dose of about 300 mg per administration, atezolizumab at a dose of
about 1200 mg per administration, and bevacizumab at a dose of
about 15 mg/kg per administration, and wherein the administration
is repeated at an interval of about 3 weeks or about 21 days
between administrations. In some embodiments, the repeated
administrations of the MOXR0916, the atezolizumab, and the
bevacizumab are administered on the same day. In some embodiments,
MOXR0916 is administered intravenously. In some embodiments,
atezolizumab is administered intravenously. In some embodiments,
bevacizumab is administered intravenously. In some embodiments,
MOXR0916, atezolizumab, and bevacizumab are administered
intravenously.
[0046] In some embodiments, provided herein is a method of treating
or delaying progression of cancer in an individual comprising
administering to the individual (i) MOXR0916 at a dose of about 160
mg, (ii) atezolizumab at a dose of about 1200 mg, and (iii)
bevacizumab at a dose of about 15 mg/kg, wherein the cancer is
selected from the group consisting of melanoma, triple-negative
breast cancer, ovarian cancer, renal cell cancer, bladder cancer,
non-small cell lung cancer, gastric cancer, and colorectal cancer.
In some embodiments, the MOXR0916, atezolizumab, and bevacizumab
are administered on the same day. In some embodiments, the method
further comprises repeating the administration of MOXR0916 at a
dose of about 160 mg per administration, atezolizumab at a dose of
about 1200 mg per administration, and bevacizumab at a dose of
about 15 mg/kg per administration, and wherein the administration
is repeated at an interval of about 3 weeks or about 21 days
between administrations. In some embodiments, the repeated
administrations of the MOXR0916, the atezolizumab, and the
bevacizumab are administered on the same day. In some embodiments,
MOXR0916 is administered intravenously. In some embodiments,
atezolizumab is administered intravenously. In some embodiments,
bevacizumab is administered intravenously. In some embodiments,
MOXR0916, atezolizumab, and bevacizumab are administered
intravenously.
[0047] In some embodiments, provided herein is a method of treating
or delaying progression of cancer in an individual comprising
administering to the individual (i) MOXR0916 at a dose of about 320
mg, (ii) atezolizumab at a dose of about 1200 mg, and (iii)
bevacizumab at a dose of about 15 mg/kg, wherein the cancer is
selected from the group consisting of melanoma, triple-negative
breast cancer, ovarian cancer, renal cell cancer, bladder cancer,
non-small cell lung cancer, gastric cancer, and colorectal cancer.
In some embodiments, the MOXR0916, atezolizumab, and bevacizumab
are administered on the same day. In some embodiments, the method
further comprises repeating the administration of MOXR0916 at a
dose of about 320 mg per administration, atezolizumab at a dose of
about 1200 mg per administration, and bevacizumab at a dose of
about 15 mg/kg per administration, and wherein the administration
is repeated at an interval of about 3 weeks or about 21 days
between administrations. In some embodiments, the repeated
administrations of the MOXR0916, the atezolizumab, and the
bevacizumab are administered on the same day. In some embodiments,
the cancer is bladder cancer. In some embodiments, MOXR0916 is
administered intravenously. In some embodiments, atezolizumab is
administered intravenously. In some embodiments, MOXR0916 and
atezolizumab are administered intravenously.
[0048] In some embodiments, provided herein is a method of treating
or delaying progression of cancer in an individual comprising
administering to the individual (i) MOXR0916 at a dose of about 400
mg, (ii) atezolizumab at a dose of about 1200 mg, and (iii)
bevacizumab at a dose of about 15 mg/kg, wherein the cancer is
selected from the group consisting of melanoma, triple-negative
breast cancer, ovarian cancer, renal cell cancer, bladder cancer,
non-small cell lung cancer, gastric cancer, and colorectal cancer.
In some embodiments, the MOXR0916, atezolizumab, and bevacizumab
are administered on the same day. In some embodiments, the method
further comprises repeating the administration of MOXR0916 at a
dose of about 400 mg per administration, atezolizumab at a dose of
about 1200 mg per administration, and bevacizumab at a dose of
about 15 mg/kg per administration, and wherein the administration
is repeated at an interval of about 3 weeks or about 21 days
between administrations. In some embodiments, the repeated
administrations of the MOXR0916, the atezolizumab, and the
bevacizumab are administered on the same day. In some embodiments,
the cancer is bladder cancer. In some embodiments, MOXR0916 is
administered intravenously. In some embodiments, atezolizumab is
administered intravenously. In some embodiments, MOXR0916 and
atezolizumab are administered intravenously.
[0049] In some embodiments of any of the above embodiments, the
methods may further comprise, after administering to the individual
the anti-human OX40 agonist antibody and the anti-PDL1 antibody,
monitoring the responsiveness of the individual to said treatment
by: (a) measuring an expression level of one or more marker genes
in a sample obtained from the cancer of the individual, wherein the
one or more marker genes are selected from the group consisting of
CCR5, CD274, IL-7, TNFRSF14, TGFB1, CD40, CD4, PRF1, TNFSF4, CD86,
CXCL9, CD3E, LAG3, PDCD1, CCL28, GZMB, IFNg, and IL-2RA; and (b)
optionally, classifying the individual as responsive or
non-responsive to treatment with the anti-human OX40 agonist
antibody and the anti-PDL1 antibody based on the expression level
of the one or more marker genes in the sample, as compared with a
reference, wherein an increased expression level of the one or more
marker genes as compared with the reference indicates a responsive
individual. In some embodiments of any of the above embodiments,
the methods may further comprise, after administering to the
individual the anti-human OX40 agonist antibody and the anti-PDL1
antibody, monitoring the responsiveness of the individual to said
treatment by: (a) measuring an expression level of one or more
marker genes in a sample obtained from the cancer of the
individual, wherein the one or more marker genes are selected from
the group consisting of CD8b, EOMES, GZMA, GZMB, IFNg, and PRF1;
and (b) optionally, classifying the individual as responsive or
non-responsive to treatment with the anti-human OX40 agonist
antibody and the anti-PDL1 antibody based on the expression level
of the one or more marker genes in the sample, as compared with a
reference, wherein an increased expression level of the one or more
marker genes as compared with the reference indicates a responsive
individual. In some embodiments of any of the above embodiments,
the methods may further comprise, after administering to the
individual the anti-human OX40 agonist antibody and the anti-PDL1
antibody, monitoring the responsiveness of the individual to said
treatment by: (a) measuring an expression level of one or more
marker genes in a sample obtained from the cancer of the
individual, wherein the one or more marker genes are selected from
the group consisting of CCL22, IL-2, RORC, IL-8, CTLA4, and FOXP3;
and (b) optionally, classifying the individual as responsive or
non-responsive to treatment with the anti-human OX40 agonist
antibody and the anti-PDL1 antibody based on the expression level
of the one or more marker genes in the sample, as compared with a
reference, wherein a decreased expression level of the one or more
marker genes as compared with the reference indicates a responsive
individual. In some embodiments of any of the above embodiments,
the methods may further comprise, after administering to the
individual the anti-human OX40 agonist antibody, the anti-PDL1
antibody, and the anti-VEGF antibody, monitoring the responsiveness
of the individual to said treatment by: (a) measuring an expression
level of one or more marker genes in a sample obtained from the
cancer of the individual, wherein the one or more marker genes are
selected from the group consisting of CCR5, CD274, IL-7, TNFRSF14,
TGFB1, CD40, CD4, PRF1, TNFSF4, CD86, CXCL9, CD3E, LAG3, PDCD1,
CCL28, GZMB, IFNg, and IL-2RA; and (b) optionally, classifying the
individual as responsive or non-responsive to treatment with the
anti-human OX40 agonist antibody, the anti-PDL1 antibody, and the
anti-VEGF antibody based on the expression level of the one or more
marker genes in the sample, as compared with a reference, wherein
an increased expression level of the one or more marker genes as
compared with the reference indicates a responsive individual. In
some embodiments of any of the above embodiments, the methods may
further comprise, after administering to the individual the
anti-human OX40 agonist antibody, the anti-PDL1 antibody, and the
anti-VEGF antibody, monitoring the responsiveness of the individual
to said treatment by: (a) measuring an expression level of one or
more marker genes in a sample obtained from the cancer of the
individual, wherein the one or more marker genes are selected from
the group consisting of CD8b, EOMES, GZMA, GZMB, IFNg, and PRF1;
and (b) optionally, classifying the individual as responsive or
non-responsive to treatment with the anti-human OX40 agonist
antibody, the anti-PDL1 antibody, and the anti-VEGF antibody based
on the expression level of the one or more marker genes in the
sample, as compared with a reference, wherein an increased
expression level of the one or more marker genes as compared with
the reference indicates a responsive individual. In some
embodiments of any of the above embodiments, the methods may
further comprise, after administering to the individual the
anti-human OX40 agonist antibody, the anti-PDL1 antibody, and the
anti-VEGF antibody, monitoring the responsiveness of the individual
to said treatment by: (a) measuring an expression level of one or
more marker genes in a sample obtained from the cancer of the
individual, wherein the one or more marker genes are selected from
the group consisting of CCL22, IL-2, RORC, IL-8, CTLA4, and FOXP3;
and (b) optionally, classifying the individual as responsive or
non-responsive to treatment with the anti-human OX40 agonist
antibody, the anti-PDL1 antibody, and the anti-VEGF antibody based
on the expression level of the one or more marker genes in the
sample, as compared with a reference, wherein a decreased
expression level of the one or more marker genes as compared with
the reference indicates a responsive individual.
[0050] In another aspect, provided herein is a method for
determining whether a cancer patient responds to a treatment with
an anti-human OX40 agonist antibody and an anti-PDL1 antibody,
comprising measuring an expression level of one or more marker
genes in a sample obtained from the cancer of the individual,
wherein the one or more marker genes are selected from the group
consisting of CCR5, CD274, IL-7, TNFRSF14, TGFB1, CD40, CD4, PRF1,
TNFSF4, CD86, CXCL9, CD3E, LAG3, PDCD1, CCL28, GZMB, IFNg, and
IL-2RA, wherein the expression level of the one or more marker
genes is compared with a reference, and wherein an increased
expression level of the one or more marker genes as compared with
the reference indicates that the cancer patient responds to said
treatment. In another aspect, provided herein is a method for
determining whether a cancer patient responds to a treatment with
an anti-human OX40 agonist antibody and an anti-PDL1 antibody,
comprising measuring an expression level of one or more marker
genes in a sample obtained from the cancer of the individual,
wherein the one or more marker genes are selected from the group
consisting of CD8b, EOMES, GZMA, GZMB, IFNg, and PRF1, wherein the
expression level of the one or more marker genes is compared with a
reference, and wherein an increased expression level of the one or
more marker genes as compared with the reference indicates that the
cancer patient responds to said treatment. In another aspect,
provided herein is a method for determining whether a cancer
patient responds to a treatment with an anti-human OX40 agonist
antibody and an anti-PDL1 antibody, comprising measuring an
expression level of one or more marker genes in a sample obtained
from the cancer of the individual, wherein the one or more marker
genes are selected from the group consisting of CCL22, IL-2, RORC,
IL-8, CTLA4, and FOXP3, wherein the expression level of the one or
more marker genes is compared with a reference, and wherein a
decreased expression level of the one or more marker genes as
compared with the reference indicates the cancer patient responds
to said treatment. In some embodiments of any of the above
embodiments, the treatment further comprises an anti-VEGF
antibody.
[0051] It is to be understood that one, some, or all of the
properties of the various embodiments described herein may be
combined to form other embodiments of the present invention. These
and other aspects of the invention will become apparent to one of
skill in the art. These and other embodiments of the invention are
further described by the detailed description that follows.
BRIEF DESCRIPTION OF THE FIGURES
[0052] FIG. 1 provides a diagram of the study design and proposed
cohorts.
[0053] FIG. 2 provides a pharmacokinetic (PK) plot of the mean
serum concentration of MOXR0916 as a function of time from first
dose for different dose groups.
[0054] FIGS. 3A-3G provide plots of peripheral OX40 receptor
occupancy at MOXR0916 doses of 0.2 mg (FIG. 3A), 3.2 mg (FIG. 3B),
12 mg (FIG. 3C), 40 mg (FIG. 3D), 80 mg (FIG. 3E), 160 mg (FIG.
3F), and 300 mg (FIG. 3G).
[0055] FIGS. 4A & 4B provide diagrams of the study design and
proposed cohorts for examining the combination of MOXR0916 and
atezolizumab (FIG. 4A), and for examining the combination of
MOXR0916, atezolizumab, and bevacizumab (FIG. 4B).
[0056] FIG. 5 shows tumor immune modulation in a biopsy of an RCC
tumor from a patient treated with MOXR0916 at a dose of 3.2 mg.
Tumor gene expression is reported as postdose fold change, relative
to predose levels.
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
I. Definitions
[0057] The term "PD-1 axis binding antagonist" refers to a molecule
that inhibits the interaction of a PD-1 axis binding partner with
either one or more of its binding partner, so as to remove T-cell
dysfunction resulting from signaling on the PD-1 signaling
axis--with a result being to restore or enhance T-cell function
(e.g., proliferation, cytokine production, target cell killing) As
used herein, a PD-1 axis binding antagonist includes a PD-1 binding
antagonist, a PD-L1 binding antagonist and a PD-L2 binding
antagonist.
[0058] The term "PD-1 binding antagonist" refers to a molecule that
decreases, blocks, inhibits, abrogates or interferes with signal
transduction resulting from the interaction of PD-1 with one or
more of its binding partners, such as PD-L1, PD-L2. In some
embodiments, the PD-1 binding antagonist is a molecule that
inhibits the binding of PD-1 to one or more of its binding
partners. In a specific aspect, the PD-1 binding antagonist
inhibits the binding of PD-1 to PD-L1 and/or PD-L2. For example,
PD-1 binding antagonists include anti-PD-1 antibodies, antigen
binding fragments thereof, immunoadhesins, fusion proteins,
oligopeptides and other molecules that decrease, block, inhibit,
abrogate or interfere with signal transduction resulting from the
interaction of PD-1 with PD-L1 and/or PD-L2. In one embodiment, a
PD-1 binding antagonist reduces the negative co-stimulatory signal
mediated by or through cell surface proteins expressed on T
lymphocytes mediated signaling through PD-1 so as render a
dysfunctional T-cell less dysfunctional (e.g., enhancing effector
responses to antigen recognition).
[0059] The term "PD-L1 binding antagonist" refers to a molecule
that decreases, blocks, inhibits, abrogates or interferes with
signal transduction resulting from the interaction of PD-L1 with
either one or more of its binding partners, such as PD-1, B7-1. In
some embodiments, a PD-L1 binding antagonist is a molecule that
inhibits the binding of PD-L1 to its binding partners. In a
specific aspect, the PD-L1 binding antagonist inhibits binding of
PD-L1 to PD-1 and/or B7-1. In some embodiments, the PD-L1 binding
antagonists include anti-PD-L1 antibodies, antigen binding
fragments thereof, immunoadhesins, fusion proteins, oligopeptides
and other molecules that decrease, block, inhibit, abrogate or
interfere with signal transduction resulting from the interaction
of PD-L1 with one or more of its binding partners, such as PD-1,
B7-1. In one embodiment, a PD-L1 binding antagonist reduces the
negative co-stimulatory signal mediated by or through cell surface
proteins expressed on T lymphocytes mediated signaling through
PD-L1 so as to render a dysfunctional T-cell less dysfunctional
(e.g., enhancing effector responses to antigen recognition). In
some embodiments, a PD-L1 binding antagonist is an anti-PD-L1
antibody. In a specific aspect, an anti-PD-L1 antibody is MPDL3280A
as described herein.
[0060] The term "PD-L2 binding antagonist" refers to a molecule
that decreases, blocks, inhibits, abrogates or interferes with
signal transduction resulting from the interaction of PD-L2 with
either one or more of its binding partners, such as PD-1. In some
embodiments, a PD-L2 binding antagonist is a molecule that inhibits
the binding of PD-L2 to one or more of its binding partners. In a
specific aspect, the PD-L2 binding antagonist inhibits binding of
PD-L2 to PD-1. In some embodiments, the PD-L2 antagonists include
anti-PD-L2 antibodies, antigen binding fragments thereof,
immunoadhesins, fusion proteins, oligopeptides and other molecules
that decrease, block, inhibit, abrogate or interfere with signal
transduction resulting from the interaction of PD-L2 with either
one or more of its binding partners, such as PD-1. In one
embodiment, a PD-L2 binding antagonist reduces the negative
co-stimulatory signal mediated by or through cell surface proteins
expressed on T lymphocytes mediated signaling through PD-L2 so as
render a dysfunctional T-cell less dysfunctional (e.g., enhancing
effector responses to antigen recognition). In some embodiments, a
PD-L2 binding antagonist is an immunoadhesin.
[0061] The term "dysfunction" in the context of immune dysfunction,
refers to a state of reduced immune responsiveness to antigenic
stimulation. The term includes the common elements of both
exhaustion and/or anergy in which antigen recognition may occur,
but the ensuing immune response is ineffective to control infection
or tumor growth.
[0062] The term "dysfunctional", as used herein, also includes
refractory or unresponsive to antigen recognition, specifically,
impaired capacity to translate antigen recognition into down-stream
T-cell effector functions, such as proliferation, cytokine
production (e.g., IL-2) and/or target cell killing.
[0063] The term "anergy" refers to the state of unresponsiveness to
antigen stimulation resulting from incomplete or insufficient
signals delivered through the T-cell receptor (e.g. increase in
intracellular Ca.sup.+2 in the absence of ras-activation). T cell
anergy can also result upon stimulation with antigen in the absence
of co-stimulation, resulting in the cell becoming refractory to
subsequent activation by the antigen even in the context of
costimulation. The unresponsive state can often be overriden by the
presence of Interleukin-2. Anergic T-cells do not undergo clonal
expansion and/or acquire effector functions.
[0064] The term "exhaustion" refers to T cell exhaustion as a state
of T cell dysfunction that arises from sustained TCR signaling that
occurs during many chronic infections and cancer. It is
distinguished from anergy in that it arises not through incomplete
or deficient signaling, but from sustained signaling. It is defined
by poor effector function, sustained expression of inhibitory
receptors and a transcriptional state distinct from that of
functional effector or memory T cells. Exhaustion prevents optimal
control of infection and tumors. Exhaustion can result from both
extrinsic negative regulatory pathways (e.g., immunoregulatory
cytokines) as well as cell intrinsic negative regulatory
(costimulatory) pathways (PD-1, B7-H3, B7-H4, etc.).
[0065] "Enhancing T cell function" means to induce, cause or
stimulate an effector or memory T cell to have a renewed, sustained
or amplified biological function. Examples of enhancing T-cell
function include: increased secretion of .gamma.-interferon from
CD8.sup.+ effector T cells, increased secretion of
.gamma.-interferon from CD4+ memory and/or effector T-cells,
increased proliferation of CD4+ effector and/or memory T cells,
increased proliferation of CD8+ effector T-cells, increased antigen
responsiveness (e.g., clearance), relative to such levels before
the intervention. In one embodiment, the level of enhancement is at
least 50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%,
200%. The manner of measuring this enhancement is known to one of
ordinary skill in the art.
[0066] A "T cell dysfunctional disorder" is a disorder or condition
of T-cells characterized by decreased responsiveness to antigenic
stimulation. In a particular embodiment, a T-cell dysfunctional
disorder is a disorder that is specifically associated with
inappropriate increased signaling through PD-1. In another
embodiment, a T-cell dysfunctional disorder is one in which T-cells
are anergic or have decreased ability to secrete cytokines,
proliferate, or execute cytolytic activity. In a specific aspect,
the decreased responsiveness results in ineffective control of a
pathogen or tumor expressing an immunogen. Examples of T cell
dysfunctional disorders characterized by T-cell dysfunction include
unresolved acute infection, chronic infection and tumor
immunity.
[0067] "Tumor immunity" refers to the process in which tumors evade
immune recognition and clearance. Thus, as a therapeutic concept,
tumor immunity is "treated" when such evasion is attenuated, and
the tumors are recognized and attacked by the immune system.
Examples of tumor recognition include tumor binding, tumor
shrinkage and tumor clearance.
[0068] "Sustained response" refers to the sustained effect on
reducing tumor growth after cessation of a treatment. For example,
the tumor size may remain to be the same or smaller as compared to
the size at the beginning of the administration phase. In some
embodiments, the sustained response has a duration at least the
same as the treatment duration, at least 1.5.times., 2.0.times.,
2.5.times., or 3.0.times. length of the treatment duration.
[0069] "Immunogenicity" refers to the ability of a particular
substance to provoke an immune response. Tumors are immunogenic and
enhancing tumor immunogenicity aids in the clearance of the tumor
cells by the immune response.
[0070] An "acceptor human framework" for the purposes herein is a
framework comprising the amino acid sequence of a light chain
variable domain (VL) framework or a heavy chain variable domain
(VH) framework derived from a human immunoglobulin framework or a
human consensus framework, as defined below. An acceptor human
framework "derived from" a human immunoglobulin framework or a
human consensus framework may comprise the same amino acid sequence
thereof, or it may contain amino acid sequence changes. In some
embodiments, the number of amino acid changes are 10 or less, 9 or
less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or
less, or 2 or less. In some embodiments, the VL acceptor human
framework is identical in sequence to the VL human immunoglobulin
framework sequence or human consensus framework sequence.
[0071] "Affinity" refers to the strength of the sum total of
noncovalent interactions between a single binding site of a
molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (Kd).
Affinity can be measured by common methods known in the art,
including those described herein. Specific illustrative and
exemplary embodiments for measuring binding affinity are described
in the following.
[0072] An "agonist antibody," as used herein, is an antibody which
activates a biological activity of the antigen it binds.
[0073] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC"
refers to a form of cytotoxicity in which secreted immunoglobulin
bound onto Fc receptors (FcRs) present on certain cytotoxic cells
(e.g. NK cells, neutrophils, and macrophages) enable these
cytotoxic effector cells to bind specifically to an antigen-bearing
target cell and subsequently kill the target cell with cytotoxins.
The primary cells for mediating ADCC, NK cells, express
Fc.gamma.RIII only, whereas monocytes express Fc.gamma.RI,
Fc.gamma.RII, and Fc.gamma.RIII. FcR expression on hematopoietic
cells is summarized in Table 3 on page 464 of Ravetch and Kinet,
Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a
molecule of interest, an in vitro ADCC assay, such as that
described in U.S. Pat. Nos. 5,500,362 or 5,821,337 or U.S. Pat. No.
6,737,056 (Presta), may be performed. Useful effector cells for
such assays include PBMC and NK cells. Alternatively, or
additionally, ADCC activity of the molecule of interest may be
assessed in vivo, e.g., in an animal model such as that disclosed
in Clynes et al. PNAS (USA) 95:652-656 (1998).
[0074] The terms "anti-OX40 antibody" and "an antibody that binds
to OX40" refer to an antibody that is capable of binding OX40 with
sufficient affinity such that the antibody is useful as a
diagnostic and/or therapeutic agent in targeting OX40. In one
embodiment, the extent of binding of an anti-OX40 antibody to an
unrelated, non-OX40 protein is less than about 10% of the binding
of the antibody to OX40 as measured, e.g., by a radioimmunoassay
(RIA). In certain embodiments, an antibody that binds to OX40 has a
dissociation constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.100 nM,
.ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM, .ltoreq.0.01 nM, or
.ltoreq.0.001 nM (e.g. 10.sup.-8M or less, e.g. from 10.sup.-8 M to
10.sup.-13 M, e.g., from 10.sup.-9 M to 10.sup.-13 M). In certain
embodiments, an anti-OX40 antibody binds to an epitope of OX40 that
is conserved among OX40 from different species.
[0075] As use herein, the term "binds", "specifically binds to" or
is "specific for" refers to measurable and reproducible
interactions such as binding between a target and an antibody,
which is determinative of the presence of the target in the
presence of a heterogeneous population of molecules including
biological molecules. For example, an antibody that binds to or
specifically binds to a target (which can be an epitope) is an
antibody that binds this target with greater affinity, avidity,
more readily, and/or with greater duration than it binds to other
targets. In one embodiment, the extent of binding of an antibody to
an unrelated target is less than about 10% of the binding of the
antibody to the target as measured, e.g., by a radioimmunoassay
(RIA). In certain embodiments, an antibody that specifically binds
to a target has a dissociation constant (Kd) of .ltoreq.1 .mu.M,
.ltoreq.100 nM, .ltoreq.10 nM, .ltoreq.1 nM, or .ltoreq.0.1 nM. In
certain embodiments, an antibody specifically binds to an epitope
on a protein that is conserved among the protein from different
species. In another embodiment, specific binding can include, but
does not require exclusive binding.
[0076] The term "antibody" herein is used in the broadest sense and
encompasses various antibody structures, including but not limited
to monoclonal antibodies, polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), and antibody fragments so
long as they exhibit the desired antigen-binding activity.
[0077] An "antibody fragment" refers to a molecule other than an
intact antibody that comprises a portion of an intact antibody that
binds the antigen to which the intact antibody binds. Examples of
antibody fragments include but are not limited to Fv, Fab, Fab',
Fab'-SH, F(ab').sub.2; diabodies; linear antibodies; single-chain
antibody molecules (e.g. scFv); and multispecific antibodies formed
from antibody fragments.
[0078] An "antibody that binds to the same epitope" as a reference
antibody refers to an antibody that blocks binding of the reference
antibody to its antigen in a competition assay by 50% or more, and
conversely, the reference antibody blocks binding of the antibody
to its antigen in a competition assay by 50% or more. An exemplary
competition assay is provided herein.
[0079] The term "binding domain" refers to the region of a
polypeptide that binds to another molecule. In the case of an FcR,
the binding domain can comprise a portion of a polypeptide chain
thereof (e.g. the alpha chain thereof) which is responsible for
binding an Fc region. One useful binding domain is the
extracellular domain of an FcR alpha chain.
[0080] A polypeptide with a variant IgG Fc with "altered" FcR, ADCC
or phagocytosis activity is one which has either enhanced or
diminished FcR binding activity (e.g, Fc.gamma.R) and/or ADCC
activity and/or phagocytosis activity compared to a parent
polypeptide or to a polypeptide comprising a native sequence Fc
region.
[0081] The term "OX40," as used herein, refers to any native OX40
from any vertebrate source, including mammals such as primates
(e.g. humans) and rodents (e.g., mice and rats), unless otherwise
indicated. The term encompasses "full-length," unprocessed OX40 as
well as any form of OX40 that results from processing in the cell.
The term also encompasses naturally occurring variants of OX40,
e.g., splice variants or allelic variants. The amino acid sequence
of an exemplary human OX40 is shown in SEQ ID NO:1.
[0082] "OX40 activation" refers to activation, of the OX40
receptor. Generally, OX40 activation results in signal
transduction.
[0083] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. Examples of cancer include but are not
limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or
lymphoid malignancies. More particular examples of such cancers
include, but not limited to, squamous cell cancer (e.g., epithelial
squamous cell cancer), lung cancer including small-cell lung
cancer, non-small cell lung cancer, adenocarcinoma of the lung and
squamous carcinoma of the lung, cancer of the peritoneum,
hepatocellular cancer, gastric or stomach cancer including
gastrointestinal cancer and gastrointestinal stromal cancer,
pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer,
liver cancer, bladder cancer, cancer of the urinary tract,
hepatoma, breast cancer, colon cancer, rectal cancer, colorectal
cancer, endometrial or uterine carcinoma, salivary gland carcinoma,
kidney or renal cancer, prostate cancer, vulval cancer, thyroid
cancer, hepatic carcinoma, anal carcinoma, penile carcinoma,
melanoma, superficial spreading melanoma, lentigo maligna melanoma,
acral lentiginous melanomas, nodular melanomas, multiple myeloma
and B-cell lymphoma; chronic lymphocytic leukemia (CLL); acute
lymphoblastic leukemia (ALL); hairy cell leukemia; chronic
myeloblastic leukemia; and post-transplant lymphoproliferative
disorder (PTLD), as well as abnormal vascular proliferation
associated with phakomatoses, edema (such as that associated with
brain tumors), Meigs' syndrome, brain, as well as head and neck
cancer, and associated metastases. In certain embodiments, cancers
that are amenable to treatment by the antibodies of the invention
include breast cancer, colorectal cancer, rectal cancer, non-small
cell lung cancer, glioblastoma, non-Hodgkins lymphoma (NHL), renal
cell cancer, prostate cancer, liver cancer, pancreatic cancer,
soft-tissue sarcoma, kaposi's sarcoma, carcinoid carcinoma, head
and neck cancer, ovarian cancer, mesothelioma, and multiple
myeloma. In some embodiments, the cancer is selected from:
non-small cell lung cancer, glioblastoma, neuroblastoma, melanoma,
breast carcinoma (e.g. triple-negative breast cancer), gastric
cancer, colorectal cancer (CRC), and hepatocellular carcinoma. Yet,
in some embodiments, the cancer is selected from: non-small cell
lung cancer, colorectal cancer, breast carcinoma (e.g.
triple-negative breast cancer), melanoma, ovarian cancer, renal
cell cancer, and bladder cancer, including metastatic forms of
those cancers. In some embodiments, the cancer is a locally
advanced or metastatic solid tumor, e.g., of any of the solid
cancers described above.
[0084] The terms "cell proliferative disorder" and "proliferative
disorder" refer to disorders that are associated with some degree
of abnormal cell proliferation. In one embodiment, the cell
proliferative disorder is cancer.
[0085] The term "chimeric" antibody refers to an antibody in which
a portion of the heavy and/or light chain is derived from a
particular source or species, while the remainder of the heavy
and/or light chain is derived from a different source or
species.
[0086] The "class" of an antibody refers to the type of constant
domain or constant region possessed by its heavy chain. There are
five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and
several of these may be further divided into subclasses (isotypes),
e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, and
IgA.sub.2. The heavy chain constant domains that correspond to the
different classes of immunoglobulins are called .alpha., .delta.,
.epsilon., .gamma., and .mu., respectively.
[0087] "Complement dependent cytotoxicity" or "CDC" refers to the
lysis of a target cell in the presence of complement. Activation of
the classical complement pathway is initiated by the binding of the
first component of the complement system (C1q) to antibodies (of
the appropriate subclass), which are bound to their cognate
antigen. To assess complement activation, a CDC assay, e.g., as
described in Gazzano-Santoro et al., J. Immunol. Methods 202:163
(1996), may be performed. Polypeptide variants with altered Fc
region amino acid sequences (polypeptides with a variant Fc region)
and increased or decreased C1q binding capability are described,
e.g., in U.S. Pat. No. 6,194,551 B1 and WO 1999/51642. See also,
e.g., Idusogie et al. J. Immunol. 164: 4178-4184 (2000).
[0088] The term "cytostatic agent" refers to a compound or
composition which arrests growth of a cell either in vitro or in
vivo. Thus, a cytostatic agent may be one which significantly
reduces the percentage of cells in S phase. Further examples of
cytostatic agents include agents that block cell cycle progression
by inducing G0/G1 arrest or M-phase arrest. The humanized anti-Her2
antibody trastuzumab (HERCEPTIN.RTM.) is an example of a cytostatic
agent that induces G0/G1 arrest. Classical M-phase blockers include
the vincas (vincristine and vinblastine), taxanes, and
topoisomerase II inhibitors such as doxorubicin, epirubicin,
daunorubicin, etoposide, and bleomycin. Certain agents that arrest
G1 also spill over into S-phase arrest, for example, DNA alkylating
agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine,
cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further
information can be found in Mendelsohn and Israel, eds., The
Molecular Basis of Cancer, Chapter 1, entitled "Cell cycle
regulation, oncogenes, and antineoplastic drugs" by Murakami et al.
(W.B. Saunders, Philadelphia, 1995), e.g., p. 13. The taxanes
(paclitaxel and docetaxel) are anticancer drugs both derived from
the yew tree. Docetaxel (TAXOTERE.RTM., Rhone-Poulenc Rorer),
derived from the European yew, is a semisynthetic analogue of
paclitaxel (TAXOL.RTM., Bristol-Myers Squibb). Paclitaxel and
docetaxel promote the assembly of microtubules from tubulin dimers
and stabilize microtubules by preventing depolymerization, which
results in the inhibition of mitosis in cells.
[0089] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents a cellular function and/or
causes cell death or destruction. Cytotoxic agents include, but are
not limited to, radioactive isotopes (e.g., At.sup.211, I.sup.131,
I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153,
Bi.sup.212, P.sup.32, Pb.sup.212 and radioactive isotopes of Lu);
chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin,
vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,
melphalan, mitomycin C, chlorambucil, daunorubicin or other
intercalating agents); growth inhibitory agents; enzymes and
fragments thereof such as nucleolytic enzymes; antibiotics; toxins
such as small molecule toxins or enzymatically active toxins of
bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof; and the various antitumor or anticancer
agents disclosed below.
[0090] A "depleting anti-OX40 antibody," is an anti-OX40 antibody
that kills or depletes OX40-expressing cells. Depletion of OX40
expressing cells can be achieved by various mechanisms, such as
antibody-dependent cell-mediated cytotoxicity and/or phagocytosis.
Depletion of OX40-expressing cells may be assayed in vitro, and
exemplary methods for in vitro ADCC and phagocytosis assays are
provided herein. In some embodiments, the OX40-expressing cell is a
human CD4+ effector T cell. In some embodiments, the
OX40-expressing cell is a transgenic BT474 cell that expresses
human OX40.
[0091] "Effector functions" refer to those biological activities
attributable to the Fc region of an antibody, which vary with the
antibody isotype. Examples of antibody effector functions include:
C1q binding and complement dependent cytotoxicity (CDC); Fc
receptor binding; antibody-dependent cell-mediated cytotoxicity
(ADCC); phagocytosis; down regulation of cell surface receptors
(e.g. B cell receptor); and B cell activation.
[0092] An "effective amount" of an agent, e.g., a pharmaceutical
formulation, refers to an amount effective, at dosages and for
periods of time necessary, to achieve the desired therapeutic or
prophylactic result.
[0093] "Fc receptor" or "FcR" describes a receptor that binds to
the Fc region of an antibody. In some embodiments, an FcR is a
native human FcR. In some embodiments, an FcR is one which binds an
IgG antibody (a gamma receptor) and includes receptors of the
Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII subclasses, including
allelic variants and alternatively spliced forms of those
receptors. Fc.gamma.RII receptors include Fc.gamma.RIIA (an
"activating receptor") and Fc.gamma.RIIB (an "inhibiting
receptor"), which have similar amino acid sequences that differ
primarily in the cytoplasmic domains thereof. Activating receptor
Fc.gamma.RIIA contains an immunoreceptor tyrosine-based activation
motif (ITAM) in its cytoplasmic domain. Inhibiting receptor
Fc.gamma.RIIB contains an immunoreceptor tyrosine-based inhibition
motif (ITIM) in its cytoplasmic domain (see, e.g., Daeron, Annu.
Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example,
in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et
al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab.
Clin. Med. 126:330-41 (1995). Other FcRs, including those to be
identified in the future, are encompassed by the term "FcR" herein.
The term "Fc receptor" or "FcR" also includes the neonatal
receptor, FcRn, which is responsible for the transfer of maternal
IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim
et al., J. Immunol. 24:249 (1994)) and regulation of homeostasis of
immunoglobulins. Methods of measuring binding to FcRn are known
(see, e.g., Ghetie and Ward., Immunol. Today 18(12):592-598 (1997);
Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton
et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004/92219
(Hinton et al.). Binding to human FcRn in vivo and serum half life
of human FcRn high affinity binding polypeptides can be assayed,
e.g., in transgenic mice or transfected human cell lines expressing
human FcRn, or in primates to which the polypeptides with a variant
Fc region are administered. WO 2000/42072 (Presta) describes
antibody variants with improved or diminished binding to FcRs. See
also, e.g., Shields et al. J. Biol. Chem. 9(2):6591-6604
(2001).
[0094] The term "Fc region" herein is used to define a C-terminal
region of an immunoglobulin heavy chain that contains at least a
portion of the constant region. The term includes native sequence
Fc regions and variant Fc regions. In one embodiment, a human IgG
heavy chain Fc region extends from Cys226, or from Pro230, to the
carboxyl-terminus of the heavy chain. However, the C-terminal
lysine (Lys447) of the Fc region may or may not be present. Unless
otherwise specified herein, numbering of amino acid residues in the
Fc region or constant region is according to the EU numbering
system, also called the EU index, as described in Kabat et al.,
Sequences of Proteins of Immunological Interest, 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.,
1991.
[0095] A "functional Fc region" possesses an "effector function" of
a native sequence Fc region. Exemplary "effector functions" include
C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; down
regulation of cell surface receptors (e.g. B cell receptor; BCR),
etc. Such effector functions generally require the Fc region to be
combined with a binding domain (e.g., an antibody variable domain)
and can be assessed using various assays as disclosed, for example,
in definitions herein.
[0096] "Human effector cells" refer to leukocytes that express one
or more FcRs and perform effector functions. In certain
embodiments, the cells express at least Fc.gamma.RIII and perform
ADCC effector function(s). Examples of human leukocytes which
mediate ADCC include peripheral blood mononuclear cells (PBMC),
natural killer (NK) cells, monocytes, cytotoxic T cells, and
neutrophils. The effector cells may be isolated from a native
source, e.g., from blood.
[0097] "Framework" or "FR" refers to variable domain residues other
than hypervariable region (HVR) residues. The FR of a variable
domain generally consists of four FR domains: FR1, FR2, FR3, and
FR4. Accordingly, the HVR and FR sequences generally appear in the
following sequence in VH (or VL):
FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
[0098] The terms "full length antibody," "intact antibody," and
"whole antibody" are used herein interchangeably to refer to an
antibody having a structure substantially similar to a native
antibody structure or having heavy chains that contain an Fc region
as defined herein.
[0099] The terms "host cell," "host cell line," and "host cell
culture" are used interchangeably and refer to cells into which
exogenous nucleic acid has been introduced, including the progeny
of such cells. Host cells include "transformants" and "transformed
cells," which include the primary transformed cell and progeny
derived therefrom without regard to the number of passages. Progeny
may not be completely identical in nucleic acid content to a parent
cell, but may contain mutations. Mutant progeny that have the same
function or biological activity as screened or selected for in the
originally transformed cell are included herein.
[0100] A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human or a human cell or derived from a non-human source that
utilizes human antibody repertoires or other human
antibody-encoding sequences. This definition of a human antibody
specifically excludes a humanized antibody comprising non-human
antigen-binding residues.
[0101] A "human consensus framework" is a framework which
represents the most commonly occurring amino acid residues in a
selection of human immunoglobulin VL or VH framework sequences.
Generally, the selection of human immunoglobulin VL or VH sequences
is from a subgroup of variable domain sequences. Generally, the
subgroup of sequences is a subgroup as in Kabat et al., Sequences
of Proteins of Immunological Interest, Fifth Edition, NIH
Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In one
embodiment, for the VL, the subgroup is subgroup kappa I as in
Kabat et al., supra. In one embodiment, for the VH, the subgroup is
subgroup III as in Kabat et al., supra.
[0102] A "humanized" antibody refers to a chimeric antibody
comprising amino acid residues from non-human HVRs and amino acid
residues from human FRs. In certain embodiments, a humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the HVRs (e.g., CDRs) correspond to those of a non-human
antibody, and all or substantially all of the FRs correspond to
those of a human antibody. A humanized antibody optionally may
comprise at least a portion of an antibody constant region derived
from a human antibody. A "humanized form" of an antibody, e.g., a
non-human antibody, refers to an antibody that has undergone
humanization.
[0103] The term "hypervariable region" or "HVR" as used herein
refers to each of the regions of an antibody variable domain which
are hypervariable in sequence ("complementarity determining
regions" or "CDRs") and/or form structurally defined loops
("hypervariable loops") and/or contain the antigen-contacting
residues ("antigen contacts"). Generally, antibodies comprise six
HVRs: three in the VH (H1, H2, H3), and three in the VL (L1, L2,
L3). Exemplary HVRs herein include:
(a) hypervariable loops occurring at amino acid residues 26-32
(L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101
(H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs
occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97
(L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al.,
Sequences of Proteins of Immunological Interest, 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.
(1991)); (c) antigen contacts occurring at amino acid residues
27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and
93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996));
and (d) combinations of (a), (b), and/or (c), including HVR amino
acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35
(H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).
[0104] Unless otherwise indicated, HVR residues and other residues
in the variable domain (e.g., FR residues) are numbered herein
according to Kabat et al., supra.
[0105] An "immunoconjugate" is an antibody conjugated to one or
more heterologous molecule(s), including but not limited to a
cytotoxic agent.
[0106] An "individual" or "subject" is a mammal. Mammals include,
but are not limited to, domesticated animals (e.g., cows, sheep,
cats, dogs, and horses), primates (e.g., humans and non-human
primates such as monkeys), rabbits, and rodents (e.g., mice and
rats). In certain embodiments, the individual or subject is a
human.
[0107] "Promoting cell growth or proliferation" means increasing a
cell's growth or proliferation by at least 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, 95%, or 100%.
[0108] An "isolated" antibody is one which has been separated from
a component of its natural environment. In some embodiments, an
antibody is purified to greater than 95% or 99% purity as
determined by, for example, electrophoretic (e.g., SDS-PAGE,
isoelectric focusing (IEF), capillary electrophoresis) or
chromatographic (e.g., ion exchange or reverse phase HPLC). For
review of methods for assessment of antibody purity, see, e.g.,
Flatman et al., J. Chromatogr. B 848:79-87 (2007).
[0109] An "isolated" nucleic acid refers to a nucleic acid molecule
that has been separated from a component of its natural
environment. An isolated nucleic acid includes a nucleic acid
molecule contained in cells that ordinarily contain the nucleic
acid molecule, but the nucleic acid molecule is present
extrachromosomally or at a chromosomal location that is different
from its natural chromosomal location.
[0110] "Isolated nucleic acid encoding an anti-OX40 antibody"
refers to one or more nucleic acid molecules encoding antibody
heavy and light chains (or fragments thereof), including such
nucleic acid molecule(s) in a single vector or separate vectors,
and such nucleic acid molecule(s) present at one or more locations
in a host cell.
[0111] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical and/or bind the same epitope, except for
possible variant antibodies, e.g., containing naturally occurring
mutations or arising during production of a monoclonal antibody
preparation, such variants generally being present in minor
amounts. In contrast to polyclonal antibody preparations, which
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody of a monoclonal
antibody preparation is directed against a single determinant on an
antigen. Thus, the modifier "monoclonal" indicates the character of
the antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by a variety of techniques, including but not
limited to the hybridoma method, recombinant DNA methods,
phage-display methods, and methods utilizing transgenic animals
containing all or part of the human immunoglobulin loci, such
methods and other exemplary methods for making monoclonal
antibodies being described herein.
[0112] A "naked antibody" refers to an antibody that is not
conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or
radiolabel. The naked antibody may be present in a pharmaceutical
formulation.
[0113] "Native antibodies" refer to naturally occurring
immunoglobulin molecules with varying structures. For example,
native IgG antibodies are heterotetrameric glycoproteins of about
150,000 daltons, composed of two identical light chains and two
identical heavy chains that are disulfide-bonded. From N- to
C-terminus, each heavy chain has a variable region (VH), also
called a variable heavy domain or a heavy chain variable domain,
followed by three constant domains (CH1, CH2, and CH3). Similarly,
from N- to C-terminus, each light chain has a variable region (VL),
also called a variable light domain or a light chain variable
domain, followed by a constant light (CL) domain. The light chain
of an antibody may be assigned to one of two types, called kappa
(.kappa.) and lambda (.lamda.), based on the amino acid sequence of
its constant domain A "native sequence Fc region" comprises an
amino acid sequence identical to the amino acid sequence of an Fc
region found in nature. Native sequence human Fc regions include a
native sequence human IgG1 Fc region (non-A and A allotypes);
native sequence human IgG2 Fc region; native sequence human IgG3 Fc
region; and native sequence human IgG4 Fc region as well as
naturally occurring variants thereof.
[0114] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, combination therapy, contraindications
and/or warnings concerning the use of such therapeutic
products.
[0115] "Percent (%) amino acid sequence identity" with respect to a
reference polypeptide sequence is defined as the percentage of
amino acid residues in a candidate sequence that are identical with
the amino acid residues in the reference polypeptide sequence,
after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering
any conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)
software. Those skilled in the art can determine appropriate
parameters for aligning sequences, including any algorithms needed
to achieve maximal alignment over the full length of the sequences
being compared. For purposes herein, however, % amino acid sequence
identity values are generated using the sequence comparison
computer program ALIGN-2. The ALIGN-2 sequence comparison computer
program was authored by Genentech, Inc., and the source code has
been filed with user documentation in the U.S. Copyright Office,
Washington D.C., 20559, where it is registered under U.S. Copyright
Registration No. TXU510087. The ALIGN-2 program is publicly
available from Genentech, Inc., South San Francisco, Calif., or may
be compiled from the source code. The ALIGN-2 program should be
compiled for use on a UNIX operating system, including digital UNIX
V4.0D. All sequence comparison parameters are set by the ALIGN-2
program and do not vary.
[0116] In situations where ALIGN-2 is employed for amino acid
sequence comparisons, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical
matches by the sequence alignment program ALIGN-2 in that program's
alignment of A and B, and where Y is the total number of amino acid
residues in B. It will be appreciated that where the length of
amino acid sequence A is not equal to the length of amino acid
sequence B, the % amino acid sequence identity of A to B will not
equal the % amino acid sequence identity of B to A. Unless
specifically stated otherwise, all % amino acid sequence identity
values used herein are obtained as described in the immediately
preceding paragraph using the ALIGN-2 computer program.
[0117] The term "pharmaceutical formulation" refers to a
preparation which is in such form as to permit the biological
activity of an active ingredient contained therein to be effective,
and which contains no additional components which are unacceptably
toxic to a subject to which the formulation would be
administered.
[0118] As used herein, "in conjunction with" refers to
administration of one treatment modality in addition to another
treatment modality. As such, "in conjunction with" refers to
administration of one treatment modality before, during, or after
administration of the other treatment modality to the
individual.
[0119] A "pharmaceutically acceptable carrier" refers to an
ingredient in a pharmaceutical formulation, other than an active
ingredient, which is nontoxic to a subject. A pharmaceutically
acceptable carrier includes, but is not limited to, a buffer,
excipient, stabilizer, or preservative.
[0120] As used herein, "treatment" (and grammatical variations
thereof such as "treat" or "treating") refers to clinical
intervention in an attempt to alter the natural course of the
individual being treated, and can be performed either for
prophylaxis or during the course of clinical pathology. Desirable
effects of treatment include, but are not limited to, preventing
occurrence or recurrence of disease, alleviation of symptoms,
diminishment of any direct or indirect pathological consequences of
the disease, preventing metastasis, decreasing the rate of disease
progression, amelioration or palliation of the disease state, and
remission or improved prognosis. In some embodiments, antibodies of
the invention are used to delay development of a disease or to slow
the progression of a disease.
[0121] The term "tumor" refers to all neoplastic cell growth and
proliferation, whether malignant or benign, and all pre-cancerous
and cancerous cells and tissues. The terms "cancer," "cancerous,"
"cell proliferative disorder," "proliferative disorder" and "tumor"
are not mutually exclusive as referred to herein.
[0122] The term "variable region" or "variable domain" refers to
the domain of an antibody heavy or light chain that is involved in
binding the antibody to antigen. The variable domains of the heavy
chain and light chain (VH and VL, respectively) of a native
antibody generally have similar structures, with each domain
comprising four conserved framework regions (FRs) and three
hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby
Immunology, 6.sup.th ed., W.H. Freeman and Co., page 91 (2007).) A
single VH or VL domain may be sufficient to confer antigen-binding
specificity. Furthermore, antibodies that bind a particular antigen
may be isolated using a VH or VL domain from an antibody that binds
the antigen to screen a library of complementary VL or VH domains,
respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887
(1993); Clarkson et al., Nature 352:624-628 (1991).
[0123] A "variant Fc region" comprises an amino acid sequence which
differs from that of a native sequence Fc region by virtue of at
least one amino acid modification, preferably one or more amino
acid substitution(s). Preferably, the variant Fc region has at
least one amino acid substitution compared to a native sequence Fc
region or to the Fc region of a parent polypeptide, e.g. from about
one to about ten amino acid substitutions, and preferably from
about one to about five amino acid substitutions in a native
sequence Fc region or in the Fc region of the parent polypeptide.
The variant Fc region herein will preferably possess at least about
80% homology with a native sequence Fc region and/or with an Fc
region of a parent polypeptide, and most preferably at least about
90% homology therewith, more preferably at least about 95% homology
therewith.
[0124] The term "vector," as used herein, refers to a nucleic acid
molecule capable of propagating another nucleic acid to which it is
linked. The term includes the vector as a self-replicating nucleic
acid structure as well as the vector incorporated into the genome
of a host cell into which it has been introduced. Certain vectors
are capable of directing the expression of nucleic acids to which
they are operatively linked Such vectors are referred to herein as
"expression vectors."
[0125] A "VH subgroup III consensus framework" comprises the
consensus sequence obtained from the amino acid sequences in
variable heavy subgroup III of Kabat et al. In one embodiment, the
VH subgroup III consensus framework amino acid sequence comprises
at least a portion or all of each of the following sequences:
EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:185)-H1-WVRQAPGKGLEWV (SEQ ID
NO:186)-H2-RFTISRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID
NO:187)-H3-WGQGTLVTVSS (SEQ ID NO:188).
[0126] A "VL subgroup I consensus framework" comprises the
consensus sequence obtained from the amino acid sequences in
variable light kappa subgroup I of Kabat et al. In one embodiment,
the VH subgroup I consensus framework amino acid sequence comprises
at least a portion or all of each of the following sequences:
DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:189)-L1-WYQQKPGKAPKLLIY (SEQ ID
NO:190)-L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID
NO:191)-L3-FGQGTKVEIK (SEQ ID NO:192).
[0127] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents a cellular function and/or
causes cell death or destruction. Cytotoxic agents include, but are
not limited to, radioactive isotopes (e.g., At211, I131, I125, Y90,
Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of
Lu); chemotherapeutic agents; growth inhibitory agents; enzymes and
fragments thereof such as nucleolytic enzymes; and toxins such as
small molecule toxins or enzymatically active toxins of bacterial,
fungal, plant or animal origin, including fragments and/or variants
thereof. Exemplary cytotoxic agents can be selected from
anti-microtubule agents, platinum coordination complexes,
alkylating agents, antibiotic agents, topoisomerase II inhibitors,
antimetabolites, topoisomerase I inhibitors, hormones and hormonal
analogues, signal transduction pathway inhibitors, non-receptor
tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents,
proapoptotic agents, inhibitors of LDH-A; inhibitors of fatty acid
biosynthesis; cell cycle signalling inhibitors; HDAC inhibitors,
proteasome inhibitors; and inhibitors of cancer metabolism.
[0128] In one embodiment the cytotoxic agent is selected from
anti-microtubule agents, platinum coordination complexes,
alkylating agents, antibiotic agents, topoisomerase II inhibitors,
antimetabolites, topoisomerase I inhibitors, hormones and hormonal
analogues, signal transduction pathway inhibitors, non-receptor
tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents,
proapoptotic agents, inhibitors of LDH-A, inhibitors of fatty acid
biosynthesis, cell cycle signalling inhibitors, HDAC inhibitors,
proteasome inhibitors, and inhibitors of cancer metabolism. In one
embodiment the cytotoxic agent is a taxane. In one embodiment the
taxane is paclitaxel or docetaxel. In one embodiment the cytotoxic
agent is a platinum agent. In one embodiment the cytotoxic agent is
an antagonist of EGFR. In one embodiment the antagonist of EGFR is
N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine
(e.g., erlotinib). In one embodiment the cytotoxic agent is a RAF
inhibitor. In one embodiment, the RAF inhibitor is a BRAF and/or
CRAF inhibitor. In one embodiment the RAF inhibitor is vemurafenib.
In one embodiment the cytotoxic agent is a PI3K inhibitor.
[0129] "Chemotherapeutic agent" includes chemical compounds useful
in the treatment of cancer. Examples of chemotherapeutic agents
include erlotinib (TARCEVA.RTM., Genentech/OSI Pharm.), bortezomib
(VELCADE.RTM., Millennium Pharm.), disulfiram, epigallocatechin
gallate, salinosporamide A, carfilzomib, 17-AAG (geldanamycin),
radicicol, lactate dehydrogenase A (LDH-A), fulvestrant
(FASLODEX.RTM., AstraZeneca), sunitib (SUTENT.RTM., Pfizer/Sugen),
letrozole (FEMARA.RTM., Novartis), imatinib mesylate (GLEEVEC.RTM.,
Novartis), finasunate (VATALANIB.RTM., Novartis), oxaliplatin
(ELOXATIN.RTM., Sanofi), 5-FU (5-fluorouracil), leucovorin,
Rapamycin (Sirolimus, RAPAMUNE.RTM., Wyeth), Lapatinib
(TYKERB.RTM., GSK572016, Glaxo Smith Kline), Lonafamib (SCH 66336),
sorafenib (NEXAVAR.RTM., Bayer Labs), gefitinib (IRESSA.RTM.,
AstraZeneca), AG1478, alkylating agents such as thiotepa and
CYTOXAN.RTM. cyclosphosphamide; alkyl sulfonates such as busulfan,
improsulfan and piposulfan; aziridines such as benzodopa,
carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines including altretamine, triethylenemelamine,
triethylenephosphoramide, triethylenethiophosphoramide and
trimethylomelamine; acetogenins (especially bullatacin and
bullatacinone); a camptothecin (including topotecan and
irinotecan); bryostatin; callystatin; CC-1065 (including its
adozelesin, carzelesin and bizelesin synthetic analogs);
cryptophycins (particularly cryptophycin 1 and cryptophycin 8);
adrenocorticosteroids (including prednisone and prednisolone);
cyproterone acetate; 5.alpha.-reductases including finasteride and
dutasteride); vorinostat, romidepsin, panobinostat, valproic acid,
mocetinostat dolastatin; aldesleukin, talc duocarmycin (including
the synthetic analogs, KW-2189 and CB1-TM1); eleutherobin;
pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards
such as chlorambucil, chlomaphazine, chlorophosphamide,
estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide
hydrochloride, melphalan, novembichin, phenesterine, prednimustine,
trofosfamide, uracil mustard; nitrosoureas such as carmustine,
chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;
antibiotics such as the enediyne antibiotics (e.g., calicheamicin,
especially calicheamicin .gamma.1I and calicheamicin .omega.1I
(Angew Chem. Intl. Ed. Engl. 1994 33:183-186); dynemicin, including
dynemicin A; bisphosphonates, such as clodronate; an esperamicin;
as well as neocarzinostatin chromophore and related chromoprotein
enediyne antibiotic chromophores), aclacinomysins, actinomycin,
authramycin, azaserine, bleomycins, cactinomycin, carabicin,
caminomycin, carzinophilin, chromomycinis, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,
ADRIAMYCIN.RTM. (doxorubicin), morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as
denopterin, methotrexate, pteropterin, trimetrexate; purine analogs
such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine;
pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine,
carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine,
floxuridine; androgens such as calusterone, dromostanolone
propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals
such as aminoglutethimide, mitotane, trilostane; folic acid
replenisher such as frolinic acid; aceglatone; aldophosphamide
glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil;
bisantrene; edatraxate; defofamine; demecolcine; diaziquone;
elfomithine; elliptinium acetate; an epothilone; etoglucid; gallium
nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as
maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamnol;
nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;
podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK.RTM.
polysaccharide complex (JHS Natural Products, Eugene, Oreg.);
razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid;
triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes
(especially T-2 toxin, verracurin A, roridin A and anguidine);
urethan; vindesine; dacarbazine; mannomustine; mitobronitol;
mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");
cyclophosphamide; thiotepa; taxoids, e.g., TAXOL (paclitaxel;
Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE.RTM.
(Cremophor-free), albumin-engineered nanoparticle formulations of
paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.),
and TAXOTERE.RTM. (docetaxel, doxetaxel; Sanofi-Aventis);
chloranmbucil; GEMZAR.RTM. (gemcitabine); 6-thioguanine;
mercaptopurine; methotrexate; platinum analogs such as cisplatin
and carboplatin; vinblastine; etoposide (VP-16); ifosfamide;
mitoxantrone; vincristine; NAVELBINE.RTM. (vinorelbine);
novantrone; teniposide; edatrexate; daunomycin; aminopterin;
capecitabine (XELODA.RTM.); ibandronate; CPT-11; topoisomerase
inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such
as retinoic acid; and pharmaceutically acceptable salts, acids and
derivatives of any of the above.
[0130] Chemotherapeutic agent also includes (i) anti-hormonal
agents that act to regulate or inhibit hormone action on tumors
such as anti-estrogens and selective estrogen receptor modulators
(SERMs), including, for example, tamoxifen (including
NOLVADEX.RTM.; tamoxifen citrate), raloxifene, droloxifene,
iodoxyfene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,
onapristone, and FARESTON.RTM. (toremifine citrate); (ii) aromatase
inhibitors that inhibit the enzyme aromatase, which regulates
estrogen production in the adrenal glands, such as, for example,
4(5)-imidazoles, aminoglutethimide, MEGASE.RTM. (megestrol
acetate), AROMASIN.RTM. (exemestane; Pfizer), formestanie,
fadrozole, RIVISOR.RTM. (vorozole), FEMARA.RTM. (letrozole;
Novartis), and ARIMIDEX.RTM. (anastrozole; AstraZeneca); (iii)
anti-androgens such as flutamide, nilutamide, bicalutamide,
leuprolide and goserelin; buserelin, tripterelin,
medroxyprogesterone acetate, diethylstilbestrol, premarin,
fluoxymesterone, all transretionic acid, fenretinide, as well as
troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv)
protein kinase inhibitors; (v) lipid kinase inhibitors; (vi)
antisense oligonucleotides, particularly those which inhibit
expression of genes in signaling pathways implicated in aberrant
cell proliferation, such as, for example, PKC-alpha, Ralf and
H-Ras; (vii) ribozymes such as VEGF expression inhibitors (e.g.,
ANGIOZYME.RTM.) and HER2 expression inhibitors; (viii) vaccines
such as gene therapy vaccines, for example, ALLOVECTIN.RTM.,
LEUVECTIN.RTM., and VAXID.RTM.; PROLEUKIN.RTM., rIL-2; a
topoisomerase 1 inhibitor such as LURTOTECAN.RTM.; ABARELIX.RTM.
rmRH; and (ix) pharmaceutically acceptable salts, acids and
derivatives of any of the above.
[0131] Chemotherapeutic agent also includes antibodies such as
alemtuzumab (Campath), bevacizumab (AVASTIN.RTM., Genentech);
cetuximab (ERBITUX.RTM., Imclone); panitumumab (VECTIBIX.RTM.,
Amgen), rituximab (RITUXAN.RTM., Genentech/Biogen Idec), pertuzumab
(OMNITARG.RTM., 2C4, Genentech), trastuzumab (HERCEPTIN.RTM.,
Genentech), tositumomab (Bexxar, Corixia), and the antibody drug
conjugate, gemtuzumab ozogamicin (MYLOTARG.RTM., Wyeth). Additional
humanized monoclonal antibodies with therapeutic potential as
agents in combination with the compounds of the invention include:
apolizumab, aselizumab, atlizumab, bapineuzumab, bivatuzumab
mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol,
cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab,
epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab
ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab,
lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab,
natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab,
omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab,
pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab,
resyvizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab,
sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab,
tefibazumab, tocilizumab, toralizumab, tucotuzumab celmoleukin,
tucusituzumab, umavizumab, urtoxazumab, ustekinumab, visilizumab,
and the anti-interleukin-12 (ABT-874/J695, Wyeth Research and
Abbott Laboratories) which is a recombinant exclusively
human-sequence, full-length IgG1 antibody genetically modified to
recognize interleukin-12 p40 protein.
[0132] Chemotherapeutic agent also includes "EGFR inhibitors,"
which refers to compounds that bind to or otherwise interact
directly with EGFR and prevent or reduce its signaling activity,
and is alternatively referred to as an "EGFR antagonist." Examples
of such agents include antibodies and small molecules that bind to
EGFR. Examples of antibodies which bind to EGFR include MAb 579
(ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL
8508), MAb 528 (ATCC CRL 8509) (see, U.S. Pat. No. 4,943,533,
Mendelsohn et al.) and variants thereof, such as chimerized 225
(C225 or Cetuximab; ERBUTIX.RTM.) and reshaped human 225 (H225)
(see, WO 96/40210, Imclone Systems Inc.); IMC-11F8, a fully human,
EGFR-targeted antibody (Imclone); antibodies that bind type II
mutant EGFR (U.S. Pat. No. 5,212,290); humanized and chimeric
antibodies that bind EGFR as described in U.S. Pat. No. 5,891,996;
and human antibodies that bind EGFR, such as ABX-EGF or Panitumumab
(see WO98/50433, Abgenix/Amgen); EMD 55900 (Stragliotto et al. Eur.
J. Cancer 32A:636-640 (1996)); EMD7200 (matuzumab) a humanized EGFR
antibody directed against EGFR that competes with both EGF and
TGF-alpha for EGFR binding (EMD/Merck); human EGFR antibody,
HuMax-EGFR (GenMab); fully human antibodies known as E1.1, E2.4,
E2.5, E6.2, E6.4, E2.11, E6.3 and E7.6.3 and described in U.S. Pat.
No. 6,235,883; MDX-447 (Medarex Inc); and mAb 806 or humanized mAb
806 (Johns et al., J. Biol. Chem. 279(29):30375-30384 (2004)). The
anti-EGFR antibody may be conjugated with a cytotoxic agent, thus
generating an immunoconjugate (see, e.g., EP659,439A2, Merck Patent
GmbH). EGFR antagonists include small molecules such as compounds
described in U.S. Pat. Nos. 5,616,582, 5,457,105, 5,475,001,
5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620,
6,596,726, 6,713,484, 5,770,599, 6,140,332, 5,866,572, 6,399,602,
6,344,459, 6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008,
and 5,747,498, as well as the following PCT publications:
WO98/14451, WO98/50038, WO99/09016, and WO99/24037. Particular
small molecule EGFR antagonists include OSI-774 (CP-358774,
erlotinib, TARCEVA.RTM. Genentech/OSI Pharmaceuticals); PD 183805
(CI 1033, 2-propenamide,
N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quin-
azolinyl]-, dihydrochloride, Pfizer Inc.); ZD1839, gefitinib
(IRESSA.RTM.)
4-(3'-Chloro-4'-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoli-
ne, AstraZeneca); ZM 105180
((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zeneca); BIBX-1382
(N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4--
d]pyrimidine-2,8-diamine, Boehringer Ingelheim); PKI-166
((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol)-
;
(R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimi-
dine); CL-387785
(N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide); EKB-569
(N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(-
dimethylamino)-2-butenamide) (Wyeth); AG1478 (Pfizer); AG1571 (SU
5271; Pfizer); dual EGFR/HER2 tyrosine kinase inhibitors such as
lapatinib (TYKERB.RTM., GSK572016 or N-[3-chloro-4-[(3
fluorophenyl)methoxy]phenyl]-6[5[[[2methylsulfonyl)ethyl]amino]methyl]-2--
furanyl]-4-quinazolinamine).
[0133] Chemotherapeutic agents also include "tyrosine kinase
inhibitors" including the EGFR-targeted drugs noted in the
preceding paragraph; small molecule HER2 tyrosine kinase inhibitor
such as TAK165 available from Takeda; CP-724,714, an oral selective
inhibitor of the ErbB2 receptor tyrosine kinase (Pfizer and OSI);
dual-HER inhibitors such as EKB-569 (available from Wyeth) which
preferentially binds EGFR but inhibits both HER2 and
EGFR-overexpressing cells; lapatinib (GSK572016; available from
Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinase inhibitor;
PKI-166 (available from Novartis); pan-HER inhibitors such as
canertinib (CI-1033; Pharmacia); Raf-1 inhibitors such as antisense
agent ISIS-5132 available from ISIS Pharmaceuticals which inhibit
Raf-1 signaling; non-HER targeted TK inhibitors such as imatinib
mesylate (GLEEVEC.RTM., available from Glaxo SmithKline);
multi-targeted tyrosine kinase inhibitors such as sunitinib
(SUTENT.RTM., available from Pfizer); VEGF receptor tyrosine kinase
inhibitors such as vatalanib (PTK787/ZK222584, available from
Novartis/Schering AG); MAPK extracellular regulated kinase I
inhibitor CI-1040 (available from Pharmacia); quinazolines, such as
PD 153035,4-(3-chloroanilino) quinazoline; pyridopyrimidines;
pyrimidopyrimidines; pyrrolopyrimidines, such as CGP 59326, CGP
60261 and CGP 62706; pyrazolopyrimidines,
4-(phenylamino)-7H-pyrrolo[2,3-d] pyrimidines; curcumin (diferuloyl
methane, 4,5-bis (4-fluoroanilino)phthalimide); tyrphostines
containing nitrothiophene moieties; PD-0183805 (Warner-Lamber);
antisense molecules (e.g. those that bind to HER-encoding nucleic
acid); quinoxalines (U.S. Pat. No. 5,804,396); tryphostins (U.S.
Pat. No. 5,804,396); ZD6474 (Astra Zeneca); PTK-787
(Novartis/Schering AG); pan-HER inhibitors such as CI-1033
(Pfizer); Affinitac (ISIS 3521; Isis/Lilly); imatinib mesylate
(GLEEVEC.RTM.); PKI 166 (Novartis); GW2016 (Glaxo SmithKline);
CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Pfizer); ZD6474
(AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11 (Imclone),
rapamycin (sirolimus, RAPAMUNE.RTM.); or as described in any of the
following patent publications: U.S. Pat. No. 5,804,396; WO
1999/09016 (American Cyanamid); WO 1998/43960 (American Cyanamid);
WO 1997/38983 (Warner Lambert); WO 1999/06378 (Warner Lambert); WO
1999/06396 (Warner Lambert); WO 1996/30347 (Pfizer, Inc); WO
1996/33978 (Zeneca); WO 1996/3397 (Zeneca) and WO 1996/33980
(Zeneca).
[0134] Chemotherapeutic agents also include dexamethasone,
interferons, colchicine, metoprine, cyclosporine, amphotericin,
metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine,
arsenic trioxide, asparaginase, BCG live, bevacuzimab, bexarotene,
cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane,
epoetin alfa, elotinib, filgrastim, histrelin acetate, ibritumomab,
interferon alfa-2a, interferon alfa-2b, lenalidomide, levamisole,
mesna, methoxsalen, nandrolone, nelarabine, nofetumomab,
oprelvekin, palifermin, pamidronate, pegademase, pegaspargase,
pegfilgrastim, pemetrexed disodium, plicamycin, porfimer sodium,
quinacrine, rasburicase, sargramostim, temozolomide, VM-26, 6-TG,
toremifene, tretinoin, ATRA, valrubicin, zoledronate, and
zoledronic acid, and pharmaceutically acceptable salts thereof.
[0135] Chemotherapeutic agents also include hydrocortisone,
hydrocortisone acetate, cortisone acetate, tixocortol pivalate,
triamcinolone acetonide, triamcinolone alcohol, mometasone,
amcinonide, budesonide, desonide, fluocinonide, fluocinolone
acetonide, betamethasone, betamethasone sodium phosphate,
dexamethasone, dexamethasone sodium phosphate, fluocortolone,
hydrocortisone-17-butyrate, hydrocortisone-17-valerate,
aclometasone dipropionate, betamethasone valerate, betamethasone
dipropionate, prednicarbate, clobetasone-17-butyrate,
clobetasol-17-propionate, fluocortolone caproate, fluocortolone
pivalate and fluprednidene acetate; immune selective
anti-inflammatory peptides (ImSAIDs) such as
phenylalanine-glutamine-glycine (FEG) and its D-isomeric form (feG)
(IMULAN BioTherapeutics, LLC); anti-rheumatic drugs such as
azathioprine, ciclosporin (cyclosporine A), D-penicillamine, gold
salts, hydroxychloroquine, leflunomideminocycline, sulfasalazine,
tumor necrosis factor alpha (TNF.alpha.) blockers such as
etanercept (Enbrel), infliximab (Remicade), adalimumab (Humira),
certolizumab pegol (Cimzia), golimumab (Simponi), Interleukin 1
(IL-1) blockers such as anakinra (Kineret), T cell costimulation
blockers such as abatacept (Orencia), Interleukin 6 (IL-6) blockers
such as tocilizumab (ACTEMERA.RTM.); Interleukin 13 (IL-13)
blockers such as lebrikizumab; Interferon alpha (IFN) blockers such
as Rontalizumab; Beta 7 integrin blockers such as rhuMAb Beta7; IgE
pathway blockers such as Anti-M1 prime; Secreted homotrimeric LTa3
and membrane bound heterotrimer LTa1/.beta.2 blockers such as
Anti-lymphotoxin alpha (LTa); radioactive isotopes (e.g., At211,
1131, 1125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and
radioactive isotopes of Lu); miscellaneous investigational agents
such as thioplatin, PS-341, phenylbutyrate, ET-18-OCH3, or farnesyl
transferase inhibitors (L-739749, L-744832); polyphenols such as
quercetin, resveratrol, piceatannol, epigallocatechine gallate,
theaflavins, flavanols, procyanidins, betulinic acid and
derivatives thereof; autophagy inhibitors such as chloroquine;
delta-9-tetrahydrocannabinol (dronabinol, MARINOL.RTM.);
beta-lapachone; lapachol; colchicines; betulinic acid;
acetylcamptothecin, scopolectin, and 9-aminocamptothecin);
podophyllotoxin; tegafur (UFTORAL.RTM.); bexarotene
(TARGRETIN.RTM.); bisphosphonates such as clodronate (for example,
BONEFOS.RTM. or OSTAC.RTM.), etidronate (DIDROCAL.RTM.), NE-58095,
zoledronic acid/zoledronate (ZOMETA.RTM.), alendronate
(FOSAMAX.RTM.), pamidronate (AREDIA.RTM.), tiludronate
(SKELID.RTM.), or risedronate (ACTONEL.RTM.); and epidermal growth
factor receptor (EGF-R); vaccines such as THERATOPE.RTM. vaccine;
perifosine, COX-2 inhibitor (e.g. celecoxib or etoricoxib),
proteosome inhibitor (e.g. PS341); CCI-779; tipifarnib (R11577);
orafenib, ABT510; Bcl-2 inhibitor such as oblimersen sodium
(GENASENSE.RTM.); pixantrone; farnesyltransferase inhibitors such
as lonafarnib (SCH 6636, SARASAR.TM.); and pharmaceutically
acceptable salts, acids or derivatives of any of the above; as well
as combinations of two or more of the above such as CHOP, an
abbreviation for a combined therapy of cyclophosphamide,
doxorubicin, vincristine, and prednisolone; and FOLFOX, an
abbreviation for a treatment regimen with oxaliplatin
(ELOXATIN.TM.) combined with 5-FU and leucovorin.
[0136] Chemotherapeutic agents also include non-steroidal
anti-inflammatory drugs with analgesic, antipyretic and
anti-inflammatory effects. NSAIDs include non-selective inhibitors
of the enzyme cyclooxygenase. Specific examples of NSAIDs include
aspirin, propionic acid derivatives such as ibuprofen, fenoprofen,
ketoprofen, flurbiprofen, oxaprozin and naproxen, acetic acid
derivatives such as indomethacin, sulindac, etodolac, diclofenac,
enolic acid derivatives such as piroxicam, meloxicam, tenoxicam,
droxicam, lornoxicam and isoxicam, fenamic acid derivatives such as
mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic
acid, and COX-2 inhibitors such as celecoxib, etoricoxib,
lumiracoxib, parecoxib, rofecoxib, rofecoxib, and valdecoxib.
NSAIDs can be indicated for the symptomatic relief of conditions
such as rheumatoid arthritis, osteoarthritis, inflammatory
arthropathies, ankylosing spondylitis, psoriatic arthritis,
Reiter's syndrome, acute gout, dysmenorrhoea, metastatic bone pain,
headache and migraine, postoperative pain, mild-to-moderate pain
due to inflammation and tissue injury, pyrexia, ileus, and renal
colic.
[0137] The term "cytokine" is a generic term for proteins released
by one cell population that act on another cell as intercellular
mediators. Examples of such cytokines are lymphokines, monokines;
interleukins (ILs) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15; a tumor necrosis
factor such as TNF-.alpha. or TNF-.beta.; and other polypeptide
factors including LIF and kit ligand (KL) and gamma interferon. As
used herein, the term cytokine includes proteins from natural
sources or from recombinant cell culture and biologically active
equivalents of the native-sequence cytokines, including
synthetically produced small-molecule entities and pharmaceutically
acceptable derivatives and salts thereof.
[0138] The term "phagocytosis" means the internalization of cells
or particulate matter by cells. In some embodiments, the phagocytic
cells or phagocytes are macrophages or neutrophils. In some
embodiments, the cells are cells that express human OX40. Methods
for assaying phagocytosis are known in the art and include use of
microscopy to detect the presence of cells internalized within
another cells. In other embodiments, phagocytosis is detected using
FACS, e.g., by detecting presence of a detectably labeled cell
within another cell (which may be detectably labeled, e.g., with a
different label than the first cell).
[0139] The phrase "does not possess substantial activity" or
"substantially no activity" with respect to an antibody, as used
herein, means the antibody does not exhibit an activity that is
above background level (in some embodiments, that is above
background level that is statistically significant). The phrase
"little to no activity" with respect to an antibody, as used
herein, means the antibody does not display a biologically
meaningful amount of a function. The function can be measured or
detected according to any assay or technique known in the art,
including, e.g., those described herein. In some embodiments,
antibody function is stimulation of effector T cell proliferation
and/or cytokine secretion.
[0140] The term "biomarker" or "marker" as used herein refers
generally to a molecule, including a gene, mRNA, protein,
carbohydrate structure, or glycolipid, the expression of which in
or on a tissue or cell or secreted can be detected by known methods
(or methods disclosed herein) and is predictive or can be used to
predict (or aid prediction) for a cell, tissue, or patient's
responsiveness to treatment regimes.
[0141] By "patient sample" is meant a collection of cells or fluids
obtained from a cancer patient. The source of the tissue or cell
sample may be solid tissue as from a fresh, frozen and/or preserved
organ or tissue sample or biopsy or aspirate; blood or any blood
constituents; bodily fluids such as cerebrospinal fluid, amniotic
fluid, peritoneal fluid, or interstitial fluid; cells from any time
in gestation or development of the subject. The tissue sample may
contain compounds which are not naturally intermixed with the
tissue in nature such as preservatives, anticoagulants, buffers,
fixatives, nutrients, antibiotics, or the like. Examples of tumor
samples herein include, but are not limited to, tumor biopsy, fine
needle aspirate, bronchiolar lavage, pleural fluid, sputum, urine,
a surgical specimen, circulating tumor cells, serum, plasma,
circulating plasma proteins, ascitic fluid, primary cell cultures
or cell lines derived from tumors or exhibiting tumor-like
properties, as well as preserved tumor samples, such as
formalin-fixed, paraffin-embedded tumor samples or frozen tumor
samples.
[0142] The phrase "based on expression of" when used herein means
that information about expression level or presence or absence of
expression (e.g., presence or absence or prevalence of (e.g.,
percentage of cells displaying) of the one or more biomarkers
herein (e.g., presence or absence of or amount or prevelance of
FcR-expressing cells, or e.g., presence or absence or amount or
prevelance of human effector cells) is used to inform a treatment
decision, information provided on a package insert, or
marketing/promotional guidance etc.
[0143] A cancer or biological sample which "has human effector
cells" is one which, in a diagnostic test, has human effector cells
present in the sample (e.g., infiltrating human effector
cells).
[0144] A cancer or biological sample which "has FcR-expressing
cells" is one which, in a diagnostic test, has FcR-expressing
present in the sample (e.g., infiltrating FcR-expressing cells). In
some embodiments, FcR is Fc.gamma.R. In some embodiments, FcR is an
activating Fc.gamma.R.
[0145] The phrase "recommending a treatment" as used herein refers
to using the information or data generated relating to the level or
presence of c-met in a sample of a patient to identify the patient
as suitably treated or not suitably treated with a therapy. In some
embodiments the therapy may comprise c-met antibody (e.g.,
onartuzumab). In some embodiments, the therapy may comprise VEGF
antagonist (e.g., bevacizumab). In some embodiments, the therapy
may comprise anti-human OX40 agonist antibody. The information or
data may be in any form, written, oral or electronic. In some
embodiments, using the information or data generated includes
communicating, presenting, reporting, storing, sending,
transferring, supplying, transmitting, delivering, dispensing, or
combinations thereof. In some embodiments, communicating,
presenting, reporting, storing, sending, transferring, supplying,
transmitting, delivering, dispensing, or combinations thereof are
performed by a computing device, analyzer unit or combination
thereof. In some further embodiments, communicating, presenting,
reporting, storing, sending, transferring, supplying, transmitting,
dispensing, or combinations thereof are performed by an individual
(e.g., a laboratory or medical professional). In some embodiments,
the information or data includes a comparison of the amount or
prevelance of FcR expressing cells to a reference level. In some
embodiments, the information or data includes a comparison of the
amount or prevelance of human effector cells to a reference level.
In some embodiments, the information or data includes an indication
that human effector cells or FcR-expressing cells are present or
absent in the sample. In some embodiments, the information or data
includes an indication that FcR-expressing cells and/or human
effector cells are present in a particular percentage of cells
(e.g., high prevelance). In some embodiments, the information or
data includes an indication that the patient is suitably treated or
not suitably treated with a therapy comprising anti-human OX40
agonist antibody.
II. Compositions and Methods
[0146] Provided herein are methods for treating or delaying
progression of cancer in an individual comprising administering to
the individual an effective amount of a PD-1 axis binding
antagonist and an OX40 binding agonist. Also provided herein are
methods of enhancing immune function in an individual having cancer
comprising administering to the individual an effective amount of a
PD-1 axis binding antagonist and an OX40 binding agonist. Without
wishing to be bound to theory, it is thought that treatment with an
OX40 binding agonist may enhance the efficacy of, or otherwise act
synergistically with, PD-1 axis binding antagonist treatment, e.g.,
through reduction in Tregs, an increase in Teff activation, and/or
an increase in PD-L1 expression, and that the complementary
mechanism of action of such agents (i.e., OX40 binding agonists and
PD-1 axis binding antagonists) support their use in combination for
treating or delaying progression of cancer and/or enhancing immune
function in an individual having cancer.
[0147] Further provided herein are methods for treating or delaying
progression of cancer in an individual comprising administering to
the individual an effective amount of a PD-1 axis binding
antagonist, an OX40 binding agonist, and an anti-angiogenesis
agent. Also provided herein are methods of enhancing immune
function in an individual having cancer comprising administering to
the individual an effective amount of a PD-1 axis binding
antagonist, an OX40 binding agonist, and an anti-angiogenesis
agent. Without wishing to be bound to theory, it is thought that
since anti-angiogenesis agents (e.g., anti-VEGF antibodies) are
thought to have immunomodulatory effects (e.g., through increasing
trafficking of T cells into tumors, reducing suppressive cytokines
and tumor-infiltrating Treg cells, and/or increasing CD8+ and CD4+
central memory T cells), combining an anti-angiogenesis agent with
a PD-1 axis binding antagonist and an OX40 binding agonist may act
synergistically to enhance the anti-tumor immune response,
particularly for (but not limited to) cancers for which
anti-angiogenesis agents are commonly administered (e.g., RCC or
CRC).
A. Exemplary Anti-OX40 Antibodies
[0148] Certain aspects of the present disclosure relate to methods
of treating or delaying progression of cancer using anti-OX40
antibodies (e.g., antibodies that bind human OX40) and PD-1 axis
binding antagonists (e.g., anti-PD-L1 antibodies). In some
embodiments, the methods of treating or delaying progression of
cancer include using anti-OX40 antibody (e.g., an antibody that
binds human OX40), PD-1 axis binding antagonist (e.g., anti-PD-L1
antibody), and an anti-angiogenesis agent (e.g., a VEGF antagonist
such as an anti-VEGF antibody).
[0149] In one aspect, the invention provides isolated antibodies
that bind to human OX40.
[0150] In some embodiments, the anti-human OX40 agonist antibody
binds human OX40 with an affinity of less than or equal to about
0.45 nM. In some embodiments, the anti-human OX40 antibody binds
human OX40 with an affinity of less than or equal to about 0.4 nM.
In some embodiments, the anti-human OX40 antibody binds human OX40
with an affinity of less than or equal to about 0.5 nM. In some
embodiments, the binding affinity is determined using
radioimmunoassay.
[0151] In some embodiments, the anti-human OX40 agonist antibody
binds human OX40 and cynomolgus OX40. In some embodiments, binding
is determined using a FACS assay. In some embodiments, binding to
human OX40 has an EC50 of about 0.2 ug/ml. In some embodiments,
binding to human OX40 has an EC50 of about 0.3 ug/ml or lower. In
some embodiments, binding to cynomolgus OX40 has an EC50 of about
1.5 ug/ml. In some embodiments, binding to cynomolgus OX40 has an
EC50 of about 1.4 ug/ml.
[0152] In some embodiments, the anti-human OX40 agonist antibody
does not bind to rat OX40 or mouse OX40.
[0153] In some embodiments, the anti-human OX40 agonist antibody is
a depleting anti-human OX40 antibody (e.g., depletes cells that
express human OX40). In some embodiments, the human OX40 expressing
cells are CD4+ effector T cells. In some embodiments, the human
OX40 expressing cells are Treg cells. In some embodiments,
depleting is by ADCC and/or phagocytosis. In some embodiments, the
antibody mediates ADCC by binding Fc.gamma.R expressed by a human
effector cell and activating the human effector cell function. In
some embodiments, the antibody mediates phagocytosis by binding
Fc.gamma.R expressed by a human effector cell and activating the
human effector cell function. Exemplary human effector cells
include, e.g., macrophage, natural killer (NK) cells, monocytes,
neutrophils. In some embodiments, the human effector cell is
macrophage. In some embodiments, the human effector cell is NK
cells. In some embodiments, depletion is not by apoptosis.
[0154] In some embodiments, the anti-human OX40 agonist antibody
has a functional Fc region. In some embodiments, effector function
of a functional Fc region is ADCC. In some embodiments, effector
function of a functional Fc region is phagocytosis. In some
embodiments, effector function of a functional Fc region is ADCC
and phagocytosis. In some embodiments, the Fc region is human IgG1.
In some embodiments, the Fc region is human IgG4.
[0155] In some embodiments, the anti-human OX40 agonist antibody
does not induce apoptosis in OX40-expressing cells (e.g., Treg). In
some embodiments, apoptosis is assayed using an antibody
concentration of 30 ug/ml, e.g., by determining whether apoptosis
has occurred using annexin V and proprodium iodide stained
Treg.
[0156] In some embodiments, the anti-human OX40 agonist antibody
enhances CD4+ effector T cell function, for example, by increasing
CD4+ effector T cell proliferation and/or increasing gamma
interferon production by the CD4+ effector T cell (for example, as
compared to proliferation and/or cytokine production prior to
treatment with anti-human OX40 agonist antibody). In some
embodiments, the cytokine is gamma interferon. In some embodiments,
the anti-human OX40 agonist antibody increases number of
intratumoral (infiltrating) CD4+ effector T cells (e.g., total
number of CD4+ effector T cells, or e.g., percentage of CD4+ cells
in CD45+ cells), e.g., as compared to number of intratumoral
(infiltrating) CD4+ T cells prior to treatment with anti-human OX40
agonist antibody. In some embodiments, the anti-human OX40 agonist
antibody increases number of intratumoral (infiltrating) CD4+
effector T cells that express gamma interferon (e.g., total gamma
interferon expressing CD4+ cells, or e.g., percentage of gamma
interferon expressing CD4+ cells in total CD4+ cells), e.g., as
compared to number of intratumoral (infiltrating) CD4+ T cells that
express gamma interferon prior to treatment with anti-human OX40
agonist antibody.
[0157] In some embodiments, the anti-human OX40 agonist antibody
increases number of intratumoral (infiltrating) CD8+ effector T
cells (e.g., total number of CD8+ effector T cells, or e.g.,
percentage of CD8+ in CD45+ cells), e.g., as compared to number of
intratumoral (infiltrating) CD8+T effector cells prior to treatment
with anti-human OX40 agonist antibody. In some embodiments, the
anti-human OX40 agonist antibody increases number of intratumoral
(infiltrating) CD8+ effector T cells that express gamma interferon
(e.g., percentage of CD8+ cells that express gamma interferon in
total CD8+ cells), e.g., compared to number of intratumoral
(infiltrating) CD8+ T cells that express gamma interferon prior to
treatment with anti-human OX40 agonist antibody.
[0158] In some embodiments, the anti-human OX40 agonist antibody
enhances memory T cell function, for example by increasing memory T
cell proliferation and/or increasing cytokine production by the
memory cell. In some embodiments, the cytokine is gamma
interferon.
[0159] In some embodiments, the anti-human OX40 agonist antibody
inhibits Treg function, for example, by decreasing Treg suppression
of effector T cell function (e.g., effector T cell proliferation
and/or effector T cell cytokine secretion). In some embodiments,
the effector T cell is a CD4+ effector T cell. In some embodiments,
the anti-human OX40 agonist antibody reduces the number of
intratumoral (infiltrating) Treg (e.g., total number of Treg or
e.g., percentage of Fox3p+ cells in CD4+ cells).
[0160] In some embodiments, the anti-human OX40 agonist antibody is
engineered to increase effector function (e.g., compared to
effector function in a wild-type IgG1). In some embodiments, the
antibody has increased binding to a Fc.gamma. receptor. In some
embodiments, the antibody lacks fucose attached (directly or
indirectly) to the Fc region. For example, the amount of fucose in
such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65%
or from 20% to 40%. In some embodiments, the Fc region comprises
bisected oligosaccharides, e.g., in which a biantennary
oligosaccharide attached to the Fc region of the antibody is
bisected by GlcNAc. In some embodiments, the antibody comprises an
Fc region with one or more amino acid substitutions which improve
ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the
Fc region (EU numbering of residues).
[0161] In some embodiments, the anti-human OX40 agonist antibody
increases OX40 signal transduction in a target cell that expresses
OX40. In some embodiments, OX40 signal transduction is detected by
monitoring NFkB downstream signaling.
[0162] In some embodiments, the anti-human OX40 agonist antibody is
stable after treatment at 40 C for two weeks.
[0163] In some embodiments, the anti-human OX40 agonist antibody
binds human effector cells, e.g., binds Fc.gamma.R (e.g., an
activating Fc.gamma.R) expressed by human effector cells. In some
embodiments, the human effector cell performs (is capable of
performing) ADCC effector function. In some embodiments, the human
effector cell performs (is capable of performing) phagocytosis
effector function.
[0164] In some embodiments, the anti-human OX40 agonist antibody
comprising a variant IgG1 Fc polypeptide comprising a mutation that
eliminates binding to human effector cells (e.g., a DANA mutation)
has diminished activity (e.g., CD4+ effector T cell function, e.g.,
proliferation), relative to anti-human OX40 agonist antibody
comprising native sequence IgG1 Fc portion. In some embodiment, the
anti-human OX40 agonist antibody comprising a variant IgG1 Fc
polypeptide comprising a mutation that eliminates binding to human
effector cells (e.g., a DANA mutation) does not possess substantial
activity (e.g., CD4+ effector T cell function, e.g.,
proliferation).
[0165] In some embodiments, antibody cross-linking is required for
anti-human OX40 agonist antibody function. In some embodiments,
function is stimulation of CD4+ effector T cell proliferation. In
some embodiments, antibody cross-linking is determined by providing
anti-human OX40 agonist antibody adhered on a solid surface (e.g.,
a cell culture plate). In some embodiments, antibody cross-linking
is determined by introducing a mutation in the antibody's IgG1 Fc
portion (e.g., a DANA mutation) and testing function of the mutant
antibody.
[0166] In some embodiments, the anti-human OX40 agonist antibody
competes for binding to human OX40 with OX40L. In some embodiments,
addition of OX40L does not enhance anti-human OX40 antibody
function in an in vitro assay.
[0167] According to another embodiment, the anti-human OX40 agonist
antibodies include any one, any combination, or all of the
following properties: (1) binds human OX40 with an affinity of less
than or equal to about 0.45 nM, in some embodiments, binds human
OX40 with an affinity of less than or equal to about 0.4 nM, in
some embodiments, binds human OX40 with an affinity of less than or
equal to about 0.5 nM, in some embodiments, the binding affinity is
determined using radioimmunoassay; (2) binds human OX40 and
cynomolgus OX40, in some embodiments, binding is determined using a
FACS assay, (3) binds human OX40 with an EC50 of about 0.2 ug/ml,
in some embodiments, binds to human OX40 has an EC50 of about 0.3
ug/ml or lower, in some embodiments, binds to cynomolgus OX40 with
an EC50 of about 1.5 ug/ml, in some embodiments, binds to
cynomolgus OX40 has an EC50 of about 1.4 ug/ml, (4) does not
substantially bind to rat OX40 or mouse OX40, (6) is a depleting
anti-human OX40 antibody (e.g., depletes cells that express human
OX40), in some embodiments, the cells are CD4+ effector T cells
and/or Treg cells, (7) enhances CD4+ effector T cell function, for
example, by increasing CD4+ effector T cell proliferation and/or
increasing gamma interferon production by the CD4+ effector T cell
(for example, as compared to proliferation and/or cytokine
production prior to treatment with anti-human OX40 agonist
antibody), (8) enhances memory T cell function, for example by
increasing memory T cell proliferation and/or increasing cytokine
production by the memory cell, (9) inhibits Treg function, for
example, by decreasing Treg suppression of effector T cell function
(e.g., effector T cell proliferation and/or effector T cell
cytokine secretion). In some embodiments, the effector T cell is a
CD4+ effector T cell, (10) increases OX40 signal transduction in a
target cell that expresses OX40 (in some embodiments, OX40 signal
transduction is detected by monitoring NFkB downstream signaling),
(11) is stable after treatment at 40 C for two weeks, (12) binds
human effector cells, e.g., binds Fc.gamma.R expressed by human
effector cells, (13) anti-human OX40 agonist antibody comprising a
variant IgG1 Fc polypeptide comprising a mutation that eliminates
binding to human effector cells (e.g., N297G) has diminished
activity (e.g., CD4+ effector T cell function, e.g.,
proliferation), relative to anti-human OX40 agonist antibody
comprising native sequence IgG1 Fc portion, in some embodiment, the
anti-human OX40 agonist antibody comprising a variant IgG1 Fc
polypeptide comprising a mutation that eliminates binding to human
effector cells (e.g., N297G) does not possess substantial activity
(e.g., CD4+ effector T cell function, e.g., proliferation), (14)
antibody cross-linking (e.g., by Fc receptor binding) is required
for anti-human OX40 agonist antibody function.
[0168] In one aspect, the invention provides an anti-human OX40
agonist antibody comprising at least one, two, three, four, five,
or six HVRs selected from (a) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:2; (b) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:3; (c) HVR-H3 comprising the amino acid
sequence of SEQ ID NO:4; (d) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:6; and (f) HVR-L3 comprising the amino acid
sequence of SEQ ID NO:7.
[0169] In one aspect, the invention provides an anti-human OX40
agonist antibody comprising at least one, at least two, or all
three VH HVR sequences selected from (a) HVR-H1 comprising the
amino acid sequence of SEQ ID NO:2; (b) HVR-H2 comprising the amino
acid sequence of SEQ ID NO:3; and (c) HVR-H3 comprising the amino
acid sequence of SEQ ID NO:4. In one embodiment, the antibody
comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:4.
In another embodiment, the antibody comprises HVR-H3 comprising the
amino acid sequence of SEQ ID NO:4 and HVR-L3 comprising the amino
acid sequence of SEQ ID NO:7. In a further embodiment, the antibody
comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:4,
HVR-L3 comprising the amino acid sequence of SEQ ID NO:7, and
HVR-H2 comprising the amino acid sequence of SEQ ID NO:3. In a
further embodiment, the antibody comprises (a) HVR-H1 comprising
the amino acid sequence of SEQ ID NO:2; (b) HVR-H2 comprising the
amino acid sequence of SEQ ID NO:3; and (c) HVR-H3 comprising the
amino acid sequence of SEQ ID NO:4.
[0170] In another aspect, the invention provides an anti-human OX40
agonist antibody comprising at least one, at least two, or all
three VL HVR sequences selected from (a) HVR-L1 comprising the
amino acid sequence of SEQ ID NO:5; (b) HVR-L2 comprising the amino
acid sequence of SEQ ID NO:6; and (c) HVR-L3 comprising the amino
acid sequence of SEQ ID NO:7. In one embodiment, the antibody
comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:5; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6;
and (c) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:7.
[0171] In another aspect, an anti-human OX40 agonist antibody of
the invention comprises (a) a VH domain comprising at least one, at
least two, or all three VH HVR sequences selected from (i) HVR-H1
comprising the amino acid sequence of SEQ ID NO:2, (ii) HVR-H2
comprising the amino acid sequence of SEQ ID NO:3, and (iii) HVR-H3
comprising an amino acid sequence selected from SEQ ID NO:4; and
(b) a VL domain comprising at least one, at least two, or all three
VL HVR sequences selected from (i) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:5, (ii) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:6, and (c) HVR-L3 comprising the amino acid
sequence of SEQ ID NO:7.
[0172] In another aspect, the invention provides an anti-human OX40
agonist antibody comprising (a) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:2; (b) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:3; (c) HVR-H3 comprising the amino acid
sequence of SEQ ID NO:4; (d) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:6; and (f) HVR-L3 comprising an amino acid
sequence selected from SEQ ID NO:7.
[0173] In one aspect, the invention provides an anti-human OX40
agonist antibody comprising at least one, two, three, four, five,
or six HVRs selected from (a) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:2; (b) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:3; (c) HVR-H3 comprising the amino acid
sequence of SEQ ID NO:4; (d) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:6; and (f) HVR-L3 comprising the amino acid
sequence of SEQ ID NO:26.
[0174] In another embodiment, the antibody comprises HVR-H3
comprising the amino acid sequence of SEQ ID NO:4 and HVR-L3
comprising the amino acid sequence of SEQ ID NO:26. In a further
embodiment, the antibody comprises HVR-H3 comprising the amino acid
sequence of SEQ ID NO:4, HVR-L3 comprising the amino acid sequence
of SEQ ID NO:26, and HVR-H2 comprising the amino acid sequence of
SEQ ID NO:3.
[0175] In another aspect, an antibody of the invention comprises
(a) a VH domain comprising at least one, at least two, or all three
VH HVR sequences selected from (i) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:2, (ii) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:3, and (iii) HVR-H3 comprising an amino acid
sequence selected from SEQ ID NO:4; and (b) a VL domain comprising
at least one, at least two, or all three VL HVR sequences selected
from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5,
(ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6, and
(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:26.
[0176] In another aspect, the invention provides an antibody
comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3;
(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; (d)
HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f)
HVR-L3 comprising an amino acid sequence selected from SEQ ID
NO:26.
[0177] In one aspect, the invention provides an anti-human OX40
agonist antibody comprising at least one, two, three, four, five,
or six HVRs selected from (a) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:2; (b) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:3; (c) HVR-H3 comprising the amino acid
sequence of SEQ ID NO:4; (d) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:6; and (f) HVR-L3 comprising the amino acid
sequence of SEQ ID NO:27.
[0178] In another embodiment, the antibody comprises HVR-H3
comprising the amino acid sequence of SEQ ID NO:4 and HVR-L3
comprising the amino acid sequence of SEQ ID NO:27. In a further
embodiment, the antibody comprises HVR-H3 comprising the amino acid
sequence of SEQ ID NO:4, HVR-L3 comprising the amino acid sequence
of SEQ ID NO:27, and HVR-H2 comprising the amino acid sequence of
SEQ ID NO:3.
[0179] In another aspect, an antibody of the invention comprises
(a) a VH domain comprising at least one, at least two, or all three
VH HVR sequences selected from (i) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:2, (ii) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:3, and (iii) HVR-H3 comprising an amino acid
sequence selected from SEQ ID NO:4; and (b) a VL domain comprising
at least one, at least two, or all three VL HVR sequences selected
from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5,
(ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6, and
(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:27.
[0180] In another aspect, the invention provides an antibody
comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3;
(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; (d)
HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f)
HVR-L3 comprising an amino acid sequence selected from SEQ ID
NO:27.
[0181] In one aspect, the invention provides an anti-human OX40
agonist antibody comprising at least one, two, three, four, five,
or six HVRs selected from (a) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:2, 8 or 9; (b) HVR-H2 comprising the amino
acid sequence of SEQ ID NO:3, 10, 11, 12, 13 or 14; (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:4, 15, or 19; (d)
HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f)
HVR-L3 comprising the amino acid sequence of SEQ ID NO:7, 22, 23,
24, 25, 26, 27, or 28.
[0182] In one aspect, the invention provides an antibody comprising
at least one, at least two, or all three VH HVR sequences selected
from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 2,
8 or 9; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:
3, 10, 11, 12, 13 or 14; and (c) HVR-H3 comprising the amino acid
sequence of SEQ ID NO: 4, 15, or 19. In one embodiment, the
antibody comprises HVR-H3 comprising the amino acid sequence of SEQ
ID NO: 4, 15, or 19. In another embodiment, the antibody comprises
HVR-H3 comprising the amino acid sequence of SEQ ID NO:4, 15, or 19
and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 7, 22,
23, 24, 25, 26, 27, or 28. In a further embodiment, the antibody
comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:
4, 15, or 19, HVR-L3 comprising the amino acid sequence of SEQ ID
NO: 7, 22, 23, 24, 25, 26, 27, or 28, and HVR-H2 comprising the
amino acid sequence of SEQ ID NO: 3, 10, 11, 12, 13 or 14. In a
further embodiment, the antibody comprises (a) HVR-H1 comprising
the amino acid sequence of SEQ ID NO: 2, 8 or 9; (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO: 3, 10, 11, 12, 13
or 14; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO: 4, 15, or 19.
[0183] In another aspect, the invention provides an antibody
comprising at least one, at least two, or all three VL HVR
sequences selected from (a) HVR-L1 comprising the amino acid
sequence of SEQ ID NO: 5; (b) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:6; and (c) HVR-L3 comprising the amino acid
sequence of SEQ ID NO: 7, 22, 23, 24, 25, 26, 27, or 28. In one
embodiment, the antibody comprises (a) HVR-L1 comprising the amino
acid sequence of SEQ ID NO:5; (b) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:6; and (c) HVR-L3 comprising the amino acid
sequence of SEQ ID NO: 7, 22, 23, 24, 25, 26, 27, or 28.
[0184] In another aspect, an antibody of the invention comprises
(a) a VH domain comprising at least one, at least two, or all three
VH HVR sequences selected from (i) HVR-H1 comprising the amino acid
sequence of SEQ ID NO: 2, 8 or 9, (ii) HVR-H2 comprising the amino
acid sequence of SEQ ID NO: 3, 10, 11, 12, 13 or 14, and (iii)
HVR-H3 comprising an amino acid sequence selected from SEQ ID NO:
4, 15, or 19; and (b) a VL domain comprising at least one, at least
two, or all three VL HVR sequences selected from (i) HVR-L1
comprising the amino acid sequence of SEQ ID NO:5, (ii) HVR-L2
comprising the amino acid sequence of SEQ ID NO:6, and (c) HVR-L3
comprising the amino acid sequence of SEQ ID NO: 7, 22, 23, 24, 25,
26, 27, or 28.
[0185] In another aspect, the invention provides an antibody
comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO: 2, 8 or 9; (b) HVR-H2 comprising the amino acid sequence of SEQ
ID NO: 3, 10, 11, 12, 13 or 14; (c) HVR-H3 comprising the amino
acid sequence of SEQ ID NO: 4, 15, or 19; (d) HVR-L1 comprising the
amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino
acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising an amino
acid sequence selected from SEQ ID NO: 7, 22, 23, 24, 25, 26, 27,
or 28.
[0186] In one aspect, the invention provides an anti-human OX40
agonist antibody comprising at least one, two, three, four, five,
or six HVRs selected from (a) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:172; (b) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:173; (c) HVR-H3 comprising the amino acid
sequence of SEQ ID NO:174; (d) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:6; and (f) HVR-L3 comprising the amino acid
sequence of SEQ ID NO:175. In some embodiment, HVR-H2 is not
DMYPDAAAASYNQKFRE (SEQ ID NO:193). In some embodiments, HVR-H3 is
not APRWAAAA (SEQ ID NO:194). In some embodiments, HVR-L3 is not
QAAAAAAAT (SEQ ID NO:195).
[0187] In one aspect, the invention provides an antibody comprising
at least one, at least two, or all three VH HVR sequences selected
from (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:172; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:173; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:174. In one embodiment, the antibody comprises HVR-H3 comprising
the amino acid sequence of SEQ ID NO:174. In another embodiment,
the antibody comprises HVR-H3 comprising the amino acid sequence of
SEQ ID NO:174 and HVR-L3 comprising the amino acid sequence of SEQ
ID NO:175. In a further embodiment, the antibody comprises HVR-H3
comprising the amino acid sequence of SEQ ID NO:174, HVR-L3
comprising the amino acid sequence of SEQ ID NO:175, and HVR-H2
comprising the amino acid sequence of SEQ ID NO:173. In a further
embodiment, the antibody comprises (a) HVR-H1 comprising the amino
acid sequence of SEQ ID NO:172; (b) HVR-H2 comprising the amino
acid sequence of SEQ ID NO:173; and (c) HVR-H3 comprising the amino
acid sequence of SEQ ID NO:174. In some embodiment, HVR-H2 is not
DMYPDAAAASYNQKFRE (SEQ ID NO:193). In some embodiments, HVR-H3 is
not APRWAAAA (SEQ ID NO:194). In some embodiments, HVR-L3 is not
QAAAAAAAT (SEQ ID NO:195).
[0188] In another aspect, the invention provides an antibody
comprising (a) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:5; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6;
and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:175.
In some embodiments, HVR-L3 is not QAAAAAAAT (SEQ ID NO:195).
[0189] In another aspect, an antibody of the invention comprises
(a) a VH domain comprising at least one, at least two, or all three
VH HVR sequences selected from (i) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:172, (ii) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:173, and (iii) HVR-H3 comprising an amino
acid sequence selected from SEQ ID NO:174; and (b) a VL domain
comprising at least one, at least two, or all three VL HVR
sequences selected from (i) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:5, (ii) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:6, and (c) HVR-L3 comprising the amino acid
sequence of SEQ ID NO:175.
[0190] In another aspect, the invention provides an antibody
comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:172; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:173; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:174; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:5; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6;
and (f) HVR-L3 comprising an amino acid sequence selected from SEQ
ID NO:175. In some embodiment, HVR-H2 is not DMYPDAAAASYNQKFRE (SEQ
ID NO:193). In some embodiments, HVR-H3 is not APRWAAAA (SEQ ID
NO:194). In some embodiments, HVR-L3 is not QAAAAAAAT (SEQ ID
NO:195).
[0191] All possible combinations of the above substitutions are
encompassed by the consensus sequences of SEQ ID NO:172, 173, 174
and 175.
[0192] In one aspect, the invention provides an anti-human OX40
agonist antibody comprising at least one, two, three, four, five,
or six HVRs selected from (a) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:29; (b) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:30; (c) HVR-H3 comprising the amino acid
sequence of SEQ ID NO:33; (d) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:37; (e) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:39; and (f) HVR-L3 comprising the amino acid
sequence of SEQ ID NO:42.
[0193] In one aspect, the invention provides an antibody comprising
at least one, at least two, or all three VH HVR sequences selected
from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29;
(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:30; and
(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33. In
one embodiment, the antibody comprises HVR-H3 comprising the amino
acid sequence of SEQ ID NO:33. In another embodiment, the antibody
comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:33
and HVR-L3 comprising the amino acid sequence of SEQ ID NO:42. In a
further embodiment, the antibody comprises HVR-H3 comprising the
amino acid sequence of SEQ ID NO:33, HVR-L3 comprising the amino
acid sequence of SEQ ID NO:42, and HVR-H2 comprising the amino acid
sequence of SEQ ID NO:30. In a further embodiment, the antibody
comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:30; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:33.
[0194] In another aspect, the invention provides an antibody
comprising at least one, at least two, or all three VL HVR
sequences selected from (a) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:37; (b) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:39; and (c) HVR-L3 comprising the amino acid
sequence of SEQ ID NO:42. In one embodiment, the antibody comprises
(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (b)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:39; and (c)
HVR-L3 comprising the amino acid sequence of SEQ ID NO:42.
[0195] In another aspect, an antibody of the invention comprises
(a) a VH domain comprising at least one, at least two, or all three
VH HVR sequences selected from (i) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:29, (ii) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:30, and (iii) HVR-H3 comprising an amino acid
sequence selected from SEQ ID NO:33; and (b) a VL domain comprising
at least one, at least two, or all three VL HVR sequences selected
from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37,
(ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:39, and
(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:42.
[0196] In another aspect, the invention provides an antibody
comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:30; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:33; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:37; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:39; and (f) HVR-L3 comprising an amino acid sequence selected
from SEQ ID NO:42.
[0197] In one aspect, the invention provides an anti-human OX40
agonist antibody comprising at least one, two, three, four, five,
or six HVRs selected from (a) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:29; (b) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:30; (c) HVR-H3 comprising the amino acid
sequence of SEQ ID NO:33; (d) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:37; (e) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:40; and (f) HVR-L3 comprising the amino acid
sequence of SEQ ID NO:42.
[0198] In another aspect, the invention provides an antibody
comprising at least one, at least two, or all three VL HVR
sequences selected from (a) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:37; (b) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:40; and (c) HVR-L3 comprising the amino acid
sequence of SEQ ID NO:42. In one embodiment, the antibody comprises
(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (b)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:40; and (c)
HVR-L3 comprising the amino acid sequence of SEQ ID NO:42.
[0199] In another aspect, an antibody of the invention comprises
(a) a VH domain comprising at least one, at least two, or all three
VH HVR sequences selected from (i) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:29, (ii) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:30, and (iii) HVR-H3 comprising an amino acid
sequence selected from SEQ ID NO:33; and (b) a VL domain comprising
at least one, at least two, or all three VL HVR sequences selected
from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37,
(ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:40, and
(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:42.
[0200] In another aspect, the invention provides an antibody
comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:30; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:33; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:37; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:40; and (f) HVR-L3 comprising an amino acid sequence selected
from SEQ ID NO:42.
[0201] In one aspect, the invention provides an anti-human OX40
agonist antibody comprising at least one, two, three, four, five,
or six HVRs selected from (a) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:29; (b) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:30, 31, or 32; (c) HVR-H3 comprising the
amino acid sequence of SEQ ID NO:33; (d) HVR-L1 comprising the
amino acid sequence of SEQ ID NO:37; (e) HVR-L2 comprising the
amino acid sequence of SEQ ID NO:39, 40 or 41; and (f) HVR-L3
comprising the amino acid sequence of SEQ ID NO:42, 43, or 44.
[0202] In one aspect, the invention provides an antibody comprising
at least one, at least two, or all three VH HVR sequences selected
from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29;
(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 30, 31,
or 32; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:33. In another embodiment, the antibody comprises HVR-H3
comprising the amino acid sequence of SEQ ID NO:33 and HVR-L3
comprising the amino acid sequence of SEQ ID NO: 42, 43, or 44. In
a further embodiment, the antibody comprises HVR-H3 comprising the
amino acid sequence of SEQ ID NO:33, HVR-L3 comprising the amino
acid sequence of SEQ ID NO: 42, 43, or 44, and HVR-H2 comprising
the amino acid sequence of SEQ ID NO: 39, 40 or 41. In a further
embodiment, the antibody comprises (a) HVR-H1 comprising the amino
acid sequence of SEQ ID NO:29; (b) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:30, 31, or 32; and (c) HVR-H3 comprising the
amino acid sequence of SEQ ID NO:33.
[0203] In another aspect, the invention provides an antibody
comprising at least one, at least two, or all three VL HVR
sequences selected from (a) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:37; (b) HVR-L2 comprising the amino acid
sequence of SEQ ID NO: 39, 40 or 41; and (c) HVR-L3 comprising the
amino acid sequence of SEQ ID NO: 42, 43, or 44. In one embodiment,
the antibody comprises (a) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:37; (b) HVR-L2 comprising the amino acid
sequence of SEQ ID NO: 39, 40 or 41; and (c) HVR-L3 comprising the
amino acid sequence of SEQ ID NO: 42, 43, or 44.
[0204] In another aspect, an antibody of the invention comprises
(a) a VH domain comprising at least one, at least two, or all three
VH HVR sequences selected from (i) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:29, (ii) HVR-H2 comprising the amino acid
sequence of SEQ ID NO: 30, 31, or 32, and (iii) HVR-H3 comprising
an amino acid sequence selected from SEQ ID NO:33; and (b) a VL
domain comprising at least one, at least two, or all three VL HVR
sequences selected from (i) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:37, (ii) HVR-L2 comprising the amino acid
sequence of SEQ ID NO: 39, 40 or 41, and (c) HVR-L3 comprising the
amino acid sequence of SEQ ID NO: 42, 43, or 44.
[0205] In another aspect, the invention provides an antibody
comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:
30, 31, or 32; (c) HVR-H3 comprising the amino acid sequence of SEQ
ID NO:33; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:37; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:
39, 40 or 41; and (f) HVR-L3 comprising an amino acid sequence
selected from SEQ ID NO: 42, 43, or 44.
[0206] In one aspect, the invention provides an anti-human OX40
agonist antibody comprising at least one, two, three, four, five,
or six HVRs selected from (a) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:29; (b) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:175; (c) HVR-H3 comprising the amino acid
sequence of SEQ ID NO:33; (d) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:37; (e) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:177; and (f) HVR-L3 comprising the amino acid
sequence of SEQ ID NO:178.
[0207] In one aspect, the invention provides an antibody comprising
at least one, at least two, or all three VH HVR sequences selected
from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29;
(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:175; and
(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33. In
another embodiment, the antibody comprises HVR-H3 comprising the
amino acid sequence of SEQ ID NO:33 and HVR-L3 comprising the amino
acid sequence of SEQ ID NO:177. In a further embodiment, the
antibody comprises HVR-H3 comprising the amino acid sequence of SEQ
ID NO:33, HVR-L3 comprising the amino acid sequence of SEQ ID
NO:178, and HVR-H2 comprising the amino acid sequence of SEQ ID
NO:176. In a further embodiment, the antibody comprises (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO:29; (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO:176; and (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:33.
[0208] In another aspect, the invention provides an antibody
comprising at least one, at least two, or all three VL HVR
sequences selected from (a) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:37; (b) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:177; and (c) HVR-L3 comprising the amino acid
sequence of SEQ ID NO:177. In one embodiment, the antibody
comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:37; (b) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:177; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:178.
[0209] In another aspect, an antibody of the invention comprises
(a) a VH domain comprising at least one, at least two, or all three
VH HVR sequences selected from (i) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:29, (ii) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:176, and (iii) HVR-H3 comprising an amino
acid sequence selected from SEQ ID NO:33; and (b) a VL domain
comprising at least one, at least two, or all three VL HVR
sequences selected from (i) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:37, (ii) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:177, and (c) HVR-L3 comprising the amino acid
sequence of SEQ ID NO:178.
[0210] In another aspect, the invention provides an antibody
comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:176; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:33; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:37; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:177; and (f) HVR-L3 comprising an amino acid sequence selected
from SEQ ID NO:178.
[0211] In any of the above embodiments, an anti-OX40 agonist
antibody is humanized.
[0212] In another aspect, an anti-human OX40 agonist antibody
comprises a heavy chain variable domain (VH) sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO:56, 58,
60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92,
94, 96, 98, 100, 108, 114, 116, 183, or 184. In certain
embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g.,
conservative substitutions), insertions, or deletions relative to
the reference sequence, but an anti-human OX40 agonist antibody
comprising that sequence retains the ability to bind to OX40. In
certain embodiments, a total of 1 to 10 amino acids have been
substituted, inserted and/or deleted in SEQ ID NO:56, 58, 60, 62,
64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,
98, 100, 108, 114, 116, 183, or 184. In certain embodiments,
substitutions, insertions, or deletions occur in regions outside
the HVRs (i.e., in the FRs). Optionally, the anti-human OX40
agonist antibody comprises the VH sequence in SEQ ID NO: SEQ ID
NO:56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,
88, 90, 92, 94, 96, 98, 100, 108, 114, 116, 183, or 184, including
post-translational modifications of that sequence. In a particular
embodiment, the VH comprises one, two or three HVRs selected from:
(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:2, (b)
HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, and (c)
HVR-H3 comprising the amino acid sequence of SEQ ID NO:4.
[0213] In another aspect, an anti-human OX40 agonist antibody is
provided, wherein the antibody comprises a light chain variable
domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to the amino acid sequence of
SEQ ID NO:57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83,
85, 87, 89, 91, 93, 95, 97, 99, 101, 109, 115 or 117. In certain
embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g.,
conservative substitutions), insertions, or deletions relative to
the reference sequence, but an anti-human OX40 agonist antibody
comprising that sequence retains the ability to bind to OX40. In
certain embodiments, a total of 1 to 10 amino acids have been
substituted, inserted and/or deleted in SEQ ID NO: 57, 59, 61, 63,
65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,
99, 101, 109, 115 or 117. In certain embodiments, the
substitutions, insertions, or deletions occur in regions outside
the HVRs (i.e., in the FRs). Optionally, the anti-human OX40
agonist antibody comprises the VL sequence in SEQ ID NO: 57, 59,
61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93,
95, 97, 99, 101, 109, 115 or 117, including post-translational
modifications of that sequence. In a particular embodiment, the VL
comprises one, two or three HVRs selected from (a) HVR-L1
comprising the amino acid sequence of SEQ ID NO:5; (b) HVR-L2
comprising the amino acid sequence of SEQ ID NO:6; and (c) HVR-L3
comprising the amino acid sequence of SEQ ID NO:7.
[0214] In another aspect, an anti-human OX40 agonist antibody
comprises a heavy chain variable domain (VH) sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO:56. In
certain embodiments, a VH sequence having at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the reference sequence, but an anti-human
OX40 agonist antibody comprising that sequence retains the ability
to bind to OX40. In certain embodiments, a total of 1 to 10 amino
acids have been substituted, inserted and/or deleted in SEQ ID
NO:56. In certain embodiments, substitutions, insertions, or
deletions occur in regions outside the HVRs (i.e., in the FRs).
Optionally, the anti-human OX40 agonist antibody comprises the VH
sequence in SEQ ID NO:56, including post-translational
modifications of that sequence. In a particular embodiment, the VH
comprises one, two or three HVRs selected from: (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO:2, (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO:3, and (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:4.
[0215] In another aspect, an anti-human OX40 agonist antibody is
provided, wherein the antibody comprises a light chain variable
domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to the amino acid sequence of
SEQ ID NO:57. In certain embodiments, a VL sequence having at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity
contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative to the reference sequence, but an
anti-human OX40 agonist antibody comprising that sequence retains
the ability to bind to OX40. In certain embodiments, a total of 1
to 10 amino acids have been substituted, inserted and/or deleted in
SEQ ID NO: 57. In certain embodiments, the substitutions,
insertions, or deletions occur in regions outside the HVRs (i.e.,
in the FRs). Optionally, the anti-human OX40 agonist antibody
comprises the VL sequence in SEQ ID NO: 57, including
post-translational modifications of that sequence. In a particular
embodiment, the VL comprises one, two or three HVRs selected from
(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (b)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (c)
HVR-L3 comprising the amino acid sequence of SEQ ID NO:7.
[0216] In another aspect, an anti-human OX40 agonist antibody
comprises a heavy chain variable domain (VH) sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO:180. In
certain embodiments, a VH sequence having at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the reference sequence, but an anti-human
OX40 agonist antibody comprising that sequence retains the ability
to bind to OX40. In certain embodiments, a total of 1 to 10 amino
acids have been substituted, inserted and/or deleted in SEQ ID
NO:180. In certain embodiments, substitutions, insertions, or
deletions occur in regions outside the HVRs (i.e., in the FRs).
Optionally, the anti-human OX40 agonist antibody comprises the VH
sequence in SEQ ID NO:180, including post-translational
modifications of that sequence. In a particular embodiment, the VH
comprises one, two or three HVRs selected from: (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO:2, (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO:3, and (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:4.
[0217] In another aspect, an anti-human OX40 agonist antibody is
provided, wherein the antibody comprises a light chain variable
domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to the amino acid sequence of
SEQ ID NO:179. In certain embodiments, a VL sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity
contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative to the reference sequence, but an
anti-human OX40 agonist antibody comprising that sequence retains
the ability to bind to OX40. In certain embodiments, a total of 1
to 10 amino acids have been substituted, inserted and/or deleted in
SEQ ID NO: 179. In certain embodiments, the substitutions,
insertions, or deletions occur in regions outside the HVRs (i.e.,
in the FRs). Optionally, the anti-human OX40 agonist antibody
comprises the VL sequence in SEQ ID NO: 179, including
post-translational modifications of that sequence. In a particular
embodiment, the VL comprises one, two or three HVRs selected from
(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (b)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (c)
HVR-L3 comprising the amino acid sequence of SEQ ID NO:7.
[0218] In another aspect, an anti-human OX40 agonist antibody
comprises a heavy chain variable domain (VH) sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO:94. In
certain embodiments, a VH sequence having at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the reference sequence, but an anti-human
OX40 agonist antibody comprising that sequence retains the ability
to bind to OX40. In certain embodiments, a total of 1 to 10 amino
acids have been substituted, inserted and/or deleted in SEQ ID
NO:94. In certain embodiments, substitutions, insertions, or
deletions occur in regions outside the HVRs (i.e., in the FRs).
Optionally, the anti-human OX40 agonist antibody comprises the VH
sequence in SEQ ID NO:94, including post-translational
modifications of that sequence. In a particular embodiment, the VH
comprises one, two or three HVRs selected from: (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO:2, (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO:3, and (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:4.
[0219] In another aspect, an anti-human OX40 agonist antibody is
provided, wherein the antibody comprises a light chain variable
domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to the amino acid sequence of
SEQ ID NO:95. In certain embodiments, a VL sequence having at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity
contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative to the reference sequence, but an
anti-human OX40 agonist antibody comprising that sequence retains
the ability to bind to OX40. In certain embodiments, a total of 1
to 10 amino acids have been substituted, inserted and/or deleted in
SEQ ID NO:95. In certain embodiments, the substitutions,
insertions, or deletions occur in regions outside the HVRs (i.e.,
in the FRs). Optionally, the anti-human OX40 agonist antibody
comprises the VL sequence in SEQ ID NO:95, including
post-translational modifications of that sequence. In a particular
embodiment, the VL comprises one, two or three HVRs selected from
(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (b)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (c)
HVR-L3 comprising the amino acid sequence of SEQ ID NO:26.
[0220] In another aspect, an anti-human OX40 agonist antibody
comprises a heavy chain variable domain (VH) sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO:96. In
certain embodiments, a VH sequence having at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the reference sequence, but an anti-human
OX40 agonist antibody comprising that sequence retains the ability
to bind to OX40. In certain embodiments, a total of 1 to 10 amino
acids have been substituted, inserted and/or deleted in SEQ ID
NO:96. In certain embodiments, substitutions, insertions, or
deletions occur in regions outside the HVRs (i.e., in the FRs).
Optionally, the anti-human OX40 agonist antibody comprises the VH
sequence in SEQ ID NO:96, including post-translational
modifications of that sequence. In a particular embodiment, the VH
comprises one, two or three HVRs selected from: (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO:2, (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO:3, and (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:4.
[0221] In another aspect, an anti-human OX40 agonist antibody is
provided, wherein the antibody comprises a light chain variable
domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to the amino acid sequence of
SEQ ID NO:97. In certain embodiments, a VL sequence having at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity
contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative to the reference sequence, but an
anti-human OX40 agonist antibody comprising that sequence retains
the ability to bind to OX40. In certain embodiments, a total of 1
to 10 amino acids have been substituted, inserted and/or deleted in
SEQ ID NO:97. In certain embodiments, the substitutions,
insertions, or deletions occur in regions outside the HVRs (i.e.,
in the FRs). Optionally, the anti-human OX40 agonist antibody
comprises the VL sequence in SEQ ID NO:97, including
post-translational modifications of that sequence. In a particular
embodiment, the VL comprises one, two or three HVRs selected from
(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (b)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (c)
HVR-L3 comprising the amino acid sequence of SEQ ID NO:27.
[0222] In another aspect, an anti-human OX40 agonist antibody
comprises a heavy chain variable domain (VH) sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 118,
120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,
146, 148. In certain embodiments, a VH sequence having at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity
contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative to the reference sequence, but an
anti-human OX40 agonist antibody comprising that sequence retains
the ability to bind to OX40. In certain embodiments, a total of 1
to 10 amino acids have been substituted, inserted and/or deleted in
SEQ ID NO: 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138,
140, 142, 144, 146, 148. In certain embodiments, substitutions,
insertions, or deletions occur in regions outside the HVRs (i.e.,
in the FRs). Optionally, the anti-human OX40 agonist antibody
comprises the VH sequence in SEQ ID NO: SEQ ID NO: 118, 120, 122,
124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148,
including post-translational modifications of that sequence. In a
particular embodiment, the VH comprises one, two or three HVRs
selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ
ID NO: 29, (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:30, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:33.
[0223] In another aspect, an anti-human OX40 agonist antibody is
provided, wherein the antibody comprises a light chain variable
domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139,
141, 143, 145, 147, 149. In certain embodiments, a VL sequence
having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative to the reference sequence, but an
anti-human OX40 agonist antibody comprising that sequence retains
the ability to bind to OX40. In certain embodiments, a total of 1
to 10 amino acids have been substituted, inserted and/or deleted in
SEQ ID NO: 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139,
141, 143, 145, 147, 149. In certain embodiments, the substitutions,
insertions, or deletions occur in regions outside the HVRs (i.e.,
in the FRs). Optionally, the anti-human OX40 agonist antibody
comprises the VL sequence in SEQ ID NO: 119, 121, 123, 125, 127,
129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, including
post-translational modifications of that sequence. In a particular
embodiment, the VL comprises one, two or three HVRs selected from
(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (b)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:39; and (c)
HVR-L3 comprising the amino acid sequence of SEQ ID NO:42.
[0224] In one embodiment, the antibody comprises the VH and VL
sequences in SEQ ID NO:56 and SEQ ID NO:57, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:58 and SEQ ID NO:59, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:60 and SEQ ID NO:61, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:62 and SEQ ID NO:63, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:64 and SEQ ID NO:65, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:66 and SEQ ID NO:67, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:68 and SEQ ID NO:69, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:70 and SEQ ID NO:71, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:72 and SEQ ID NO:73, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:74 and SEQ ID NO:75, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:76 and SEQ ID NO:77, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:78 and SEQ ID NO:79, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:80 and SEQ ID NO:81, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:82 and SEQ ID NO:83, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:84 and SEQ ID NO:85, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:86 and SEQ ID NO:87, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:88 and SEQ ID NO:89, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:90 and SEQ ID NO:91, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:92 and SEQ ID NO:93, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:94 and SEQ ID NO:95, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:96 and SEQ ID NO:97, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:98 and SEQ ID NO:99, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:100 and SEQ ID NO:101, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:108 and SEQ ID NO:109, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:114 and SEQ ID NO:115, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:116 and SEQ ID NO:117, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:183 and SEQ ID NO:65, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:184 and SEQ ID NO:69, respectively, including
post-translational modifications of those sequences.
[0225] In one embodiment, the antibody comprises the VH and VL
sequences in SEQ ID NO:118 and SEQ ID NO:119, respectively,
including post-translational modifications of those sequences. In
one embodiment, the antibody comprises the VH and VL sequences in
SEQ ID NO:120 and SEQ ID NO:121, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:122 and SEQ ID NO:123, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:124 and SEQ ID NO:125, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:126 and SEQ ID NO:127, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:128 and SEQ ID NO:129, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:130 and SEQ ID NO:131, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:132 and SEQ ID NO:133, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:134 and SEQ ID NO:135, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:136 and SEQ ID NO:137, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:138 and SEQ ID NO:139, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:140 and SEQ ID NO:141, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:142 and SEQ ID NO:143, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:144 and SEQ ID NO:145, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:146 and SEQ ID NO:147, respectively, including
post-translational modifications of those sequences.
[0226] In another aspect, an anti-human OX40 agonist antibody is
provided, wherein the antibody comprises a VH as in any of the
embodiments provided above, and a VL as in any of the embodiments
provided above.
[0227] In a further aspect, the invention provides an antibody that
binds to the same epitope as an anti-human OX40 antibody provided
herein. In some embodiments, the antibody is an anti-human OX40
agonist antibody.
[0228] In a further aspect of the invention, an anti-OX40 antibody
according to any of the above embodiments is a monoclonal antibody,
including a chimeric, humanized or human antibody. In one
embodiment, an anti-OX40 antibody is an antibody fragment, e.g., a
Fv, Fab, Fab', scFv, diabody, or F(ab').sub.2 fragment. In another
embodiment, the antibody is a full length antibody, e.g., an intact
IgG1 antibody or other antibody class or isotype as defined herein.
In some embodiments, the antibody is a full length intact IgG4
antibody.
B. Exemplary PD-1 Axis Binding Antagonists
[0229] Certain aspects of the present disclosure relate to methods
of treating or delaying progression of cancer using PD-1 axis
binding antagonists (e.g., anti-PD-L1 antibodies) and anti-OX40
antibodies (e.g., antibodies that bind human OX40). In some
embodiments, the methods of treating or delaying progression of
cancer include using a PD-1 axis binding antagonist (e.g.,
anti-PD-L1 antibody), an anti-OX40 antibody (e.g., an antibody that
binds human OX40), and an anti-angiogenesis agent (e.g., a VEGF
antagonist such as an anti-VEGF antibody).
[0230] Certain aspects of the present disclosure relate to PD-1
axis binding antagonists. For example, a PD-1 axis binding
antagonist includes a PD-1 binding antagonist, a PDL1 binding
antagonist and a PDL2 binding antagonist. Alternative names for
"PD-1" include CD279 and SLEB2. Alternative names for "PDL1"
include B7-H1, B7-4, CD274, and B7-H. Alternative names for "PDL2"
include B7-DC, Btdc, and CD273. In some embodiments, PD-1, PDL1,
and PDL2 are human PD-1, PDL1 and PDL2. In some embodiments, the
PD-1 axis binding antagonist is a PDL1 binding antagonist, e.g., an
anti-PDL1 antibody.
[0231] In some embodiments, the PD-1 binding antagonist is a
molecule that inhibits the binding of PD-1 to its ligand binding
partners. In a specific aspect the PD-1 ligand binding partners are
PDL1 and/or PDL2. In another embodiment, a PDL1 binding antagonist
is a molecule that inhibits the binding of PDL1 to its binding
partners. In a specific aspect, PDL1 binding partners are PD-1
and/or B7-1. In another embodiment, the PDL2 binding antagonist is
a molecule that inhibits the binding of PDL2 to its binding
partners. In a specific aspect, a PDL2 binding partner is PD-1. The
antagonist may be an antibody, an antigen binding fragment thereof,
an immunoadhesin, a fusion protein, or oligopeptide.
[0232] In some embodiments, the PD-1 binding antagonist is an
anti-PD-1 antibody (e.g., a human antibody, a humanized antibody,
or a chimeric antibody). In some embodiments, the anti-PD-1
antibody is selected from the group consisting of MDX-1106
(nivolumab, OPDIVO), Merck 3475 (MK-3475, pembrolizumab, KEYTRUDA),
CT-011 (Pidilizumab or MDV9300), MEDI-0680 (AMP-514), PDR001,
REGN2810, BGB-108, and BGB-A317. In some embodiments, the PD-1
binding antagonist is an immunoadhesin (e.g., an immunoadhesin
comprising an extracellular or PD-1 binding portion of PDL1 or PDL2
fused to a constant region (e.g., an Fc region of an immunoglobulin
sequence). In some embodiments, the PD-1 binding antagonist is
AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538,
BMS-936558, and OPDIVO.RTM., is an anti-PD-1 antibody described in
WO2006/121168. Pembrolizumab, also known as MK-3475, Merck 3475,
lambrolizumab, KEYTRUDA.RTM., and SCH-900475, is an anti-PD-1
antibody described in WO2009/114335. CT-011, also known as hBAT or
hBAT-1, is an anti-PD-1 antibody described in WO2009/101611.
AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble
receptor described in WO2010/027827 and WO2011/066342.
[0233] In some embodiments, the anti-PD-1 antibody is nivolumab
(CAS Registry Number: 946414-94-4). In a still further embodiment,
provided is an isolated anti-PD-1 antibody comprising a heavy chain
variable region comprising the heavy chain variable region amino
acid sequence from SEQ ID NO:210 and/or a light chain variable
region comprising the light chain variable region amino acid
sequence from SEQ ID NO:211. In a still further embodiment,
provided is an isolated anti-PD-1 antibody comprising a heavy chain
and/or a light chain sequence, wherein: [0234] (a) the heavy chain
sequence has at least 85%, at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99% or 100% sequence identity to
the heavy chain sequence:
TABLE-US-00001 [0234] (SEQ ID NO: 210)
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAV
IWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATND
DYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPV
TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH
KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTP
EVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLT
VLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEE
MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
or [0235] (b) the light chain sequences has at least 85%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%
or 100% sequence identity to the light chain sequence:
TABLE-US-00002 [0235] (SEQ ID NO: 211)
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYD
ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC.
[0236] In some embodiments, the anti-PD-1 antibody is pembrolizumab
(CAS Registry Number: 1374853-91-4). In a still further embodiment,
provided is an isolated anti-PD-1 antibody comprising a heavy chain
variable region comprising the heavy chain variable region amino
acid sequence from SEQ ID NO:212 and/or a light chain variable
region comprising the light chain variable region amino acid
sequence from SEQ ID NO:213. In a still further embodiment,
provided is an isolated anti-PD-1 antibody comprising a heavy chain
and/or a light chain sequence, wherein: [0237] (a) the heavy chain
sequence has at least 85%, at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99% or 100% sequence identity to
the heavy chain sequence:
TABLE-US-00003 [0237] (SEQ ID NO: 212) QVQLVQSGVE VKKPGASVKV
SCKASGYTFT NYYMYWVRQA PGQGLEWMGG INPSNGGTNF NEKFKNRVTL TTDSSTTTAY
MELKSLQFDD TAVYYCARRDYRFDMGFDYW GQGTTVTVSS ASTKGPSVFP LAPCSRSTSE
STAALGCLVKDYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT
VPSSSLGTKTYTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV
FLFPPKPKDTLMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK
PREEQFNSTYRVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK
GQPREPQVYTLPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN
YKTTPPVLDSDGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK
or [0238] (b) the light chain sequences has at least 85%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%
or 100% sequence identity to the light chain sequence:
TABLE-US-00004 [0238] (SEQ ID NO: 213) EIVLTQSPAT
LSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAP RL LIYLASYLES GVPARFSGSG
SGTDFTLTISSLEPEDFAVYYCQHS RDLP LTFGGGTKVEI KRTVAAPSVF IFPPSDEQLK
SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC.
[0239] In some embodiments, the PDL1 binding antagonist is
anti-PDL1 antibody. In some embodiments, the anti-PDL1 binding
antagonist is selected from the group consisting of YW243.55.570,
MPDL3280A (atezolizumab), MDX-1105, MEDI4736 (durvalumab), and
MSB0010718C (avelumab). MDX-1105, also known as BMS-936559, is an
anti-PDL1 antibody described in WO2007/005874. Antibody
YW243.55.570 (heavy and light chain variable region sequences shown
in SEQ ID Nos. 20 and 21, respectively) is an anti-PDL1 described
in WO 2010/077634 A1. MEDI4736 is an anti-PDL1 antibody described
in WO2011/066389 and US2013/034559.
[0240] Examples of anti-PDL1 antibodies useful for the methods of
this invention, and methods for making thereof are described in PCT
patent application WO 2010/077634 A1 and U.S. Pat. No. 8,217,149,
which are incorporated herein by reference.
[0241] In some embodiments, the PD-1 axis binding antagonist is an
anti-PDL1 antibody. In some embodiments, the anti-PDL1 antibody is
capable of inhibiting binding between PDL1 and PD-1 and/or between
PDL1 and B7-1. In some embodiments, the anti-PDL1 antibody is a
monoclonal antibody. In some embodiments, the anti-PDL1 antibody is
an antibody fragment selected from the group consisting of Fab,
Fab'-SH, Fv, scFv, and (Fab').sub.2 fragments. In some embodiments,
the anti-PDLL antibody is a humanized antibody. In some
embodiments, the anti-PDL1 antibody is a human antibody.
[0242] The anti-PDL1 antibodies useful in this invention, including
compositions containing such antibodies, such as those described in
WO 2010/077634 A1, may be used in combination with an OX40 binding
agonist to treat cancer. In some embodiments, the anti-PDL1
antibody comprises a heavy chain variable region comprising the
amino acid sequence of SEQ ID NO:202 or 203 and a light chain
variable region comprising the amino acid sequence of SEQ ID
NO:204.
[0243] In another aspect, the polypeptide further comprises
variable region heavy chain framework sequences juxtaposed between
the HVRs according to the formula:
(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4). In
yet another aspect, the framework sequences are derived from human
consensus framework sequences. In a further aspect, the framework
sequences are VH subgroup III consensus framework. In a still
further aspect, at least one of the framework sequences is the
following:
TABLE-US-00005 (SEQ ID NO: 185) HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS
(SEQ ID NO: 186) HC-FR2 is WVRQAPGKGLEWV (SEQ ID NO: 207) HC-FR3 is
RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 208) HC-FR4 is
WGQGTLVTVSA.
[0244] In another aspect, the heavy chain variable region comprises
one or more framework sequences juxtaposed between the HVRs as:
(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and
the light chain variable regions comprises one or more framework
sequences juxtaposed between the HVRs as:
(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In
yet another aspect, the framework sequences are derived from human
consensus framework sequences. In a further aspect, the heavy chain
framework sequences are derived from a Kabat subgroup I, II, or III
sequence. In a still further aspect, the heavy chain framework
sequence is a VH subgroup III consensus framework. In a still
further aspect, one or more of the heavy chain framework sequences
is the following:
TABLE-US-00006 HC-FR1 (SEQ ID NO: 185) EVQLVESGGGLVQPGGSLRLSCAAS
HC-FR2 (SEQ ID NO: 186) WVRQAPGKGLEWV HC-FR3 (SEQ ID NO: 207)
RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR HC-FR4 (SEQ ID NO: 188)
WGQGTLVTVSS.
[0245] In a still further aspect, the light chain framework
sequences are derived from a Kabat kappa I, II, II or IV subgroup
sequence. In a still further aspect, the light chain framework
sequences are VL kappa I consensus framework. In a still further
aspect, one or more of the light chain framework sequences is the
following:
TABLE-US-00007 LC-FR1 (SEQ ID NO: 189) DIQMTQSPSSLSASVGDRVTITC
LC-FR2 (SEQ ID NO: 190) WYQQKPGKAPKLLIY LC-FR3 (SEQ ID NO: 191)
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LC-FR4 (SEQ ID NO: 208)
FGQGTKVEIKR.
[0246] In a still further specific aspect, the anti-PDL1 antibody
further comprises a human or murine constant region. In a still
further aspect, the human constant region is selected from the
group consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In a still
further specific aspect, the human constant region is IgG1. In a
still further aspect, the murine constant region is selected from
the group consisting of IgG1, IgG2A, IgG2B, IgG3. In a still
further aspect, the murine constant region if IgG2A. In a still
further specific aspect, the anti-PDL1 antibody has reduced or
minimal effector function. In a still further specific aspect the
minimal effector function results from an "effector-less Fc
mutation" or aglycosylation. In still a further embodiment, the
effector-less Fc mutation is an N297A or D265A/N297A substitution
in the constant region.
[0247] In yet another embodiment, provided is an anti-PDL1 antibody
comprising a heavy chain and a light chain variable region
sequence, wherein: [0248] (a) the heavy chain further comprises and
HVR-H1, HVR-H2 and an HVR-H3 sequence having at least 85% sequence
identity to GFTFSDSWIH (SEQ ID NO:196), AWISPYGGSTYYADSVKG (SEQ ID
NO:197) and RHWPGGFDY (SEQ ID NO:198), respectively, or [0249] (b)
the light chain further comprises an HVR-L1, HVR-L2 and an HVR-L3
sequence having at least 85% sequence identity to RASQDVSTAVA (SEQ
ID NO:199), SASFLYS (SEQ ID NO:200) and QQYLYHPAT (SEQ ID NO:201),
respectively.
[0250] In a still further embodiment, provided is an isolated
anti-PDL1 antibody comprising a heavy chain and a light chain
variable region sequence, wherein: [0251] (a) the heavy chain
sequence has at least 85% sequence identity to the heavy chain
sequence:
TABLE-US-00008 [0251] (SEQ ID NO: 209)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAW
ISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRH
WPGGFDYWGQGTLVTVSA,
or [0252] (b) the light chain sequence has at least 85% sequence
identity to the light chain sequence:
TABLE-US-00009 [0252] (SEQ ID NO: 204)
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIY SASF
LYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPAT FGQGTKVEIKR.
[0253] In another further embodiment, provided is an isolated
anti-PDL1 antibody comprising a heavy chain and a light chain
variable region sequence, wherein: [0254] (a) the heavy chain
sequence has at least 85% sequence identity to the heavy chain
sequence:
TABLE-US-00010 [0254] (SEQ ID NO: 202)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAW
ISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRH
WPGGFDYWGQGTLVTVSS,
or [0255] (b) the light chain sequence has at least 85% sequence
identity to the light chain sequence:
TABLE-US-00011 [0255] (SEQ ID NO: 204)
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIY SASF
LYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPAT FGQGTKVEIKR.
[0256] In a still further embodiment, provided is an isolated
anti-PDL1 antibody comprising a heavy chain and a light chain
variable region sequence, wherein: [0257] (a) the heavy chain
sequence has at least 85% sequence identity to the heavy chain
sequence:
TABLE-US-00012 [0257] (SEQ ID NO: 203)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWI
SPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWP
GGFDYWGQGTLVTVSSASTK,
or [0258] (b) the light chain sequences has at least 85% sequence
identity to the light chain sequence:
TABLE-US-00013 [0258] (SEQ ID NO: 204)
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSA
SFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGT KVEIKR.
[0259] In yet another embodiment, the anti-PDL1 antibody is
MPDL3280A (CAS Registry Number: 1422185-06-5). In a still further
embodiment, provided is an isolated anti-PDL1 antibody comprising a
heavy chain variable region comprising the heavy chain variable
region amino acid sequence from
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADS
VKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID
NO:202) or EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWI
SPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTL
VTVSSASTK (SEQ ID NO:203) and a light chain variable region
comprising the amino acid sequence of
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIY SASF
LYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID
NO:204).
[0260] In a still further embodiment, provided is an isolated
anti-PDL1 antibody comprising a heavy chain and/or a light chain
sequence, wherein: [0261] (a) the heavy chain sequence has at least
85%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99% or 100% sequence identity to the heavy chain
sequence:
TABLE-US-00014 [0261] (SEQ ID NO: 205)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAW
ISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRH
WPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY
FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI
CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD
TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYAST
YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY
TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG,
and/or [0262] (b) the light chain sequences has at least 85%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% or 100% sequence identity to the light chain sequence:
TABLE-US-00015 [0262] (SEQ ID NO: 206)
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIY
SASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATF
GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV
THQGLSSPVTKSFNRGEC.
[0263] In a still further embodiment, the invention provides for
compositions comprising any of the above described anti-PDL1
antibodies in combination with at least one
pharmaceutically-acceptable carrier.
[0264] In some embodiments, the isolated anti-PDL1 antibody is
aglycosylated. Glycosylation of antibodies is typically either
N-linked or O-linked N-linked refers to the attachment of the
carbohydrate moiety to the side chain of an asparagine residue. The
tripeptide sequences asparagine-X-serine and
asparagine-X-threonine, where X is any amino acid except proline,
are the recognition sequences for enzymatic attachment of the
carbohydrate moiety to the asparagine side chain. Thus, the
presence of either of these tripeptide sequences in a polypeptide
creates a potential glycosylation site. O-linked glycosylation
refers to the attachment of one of the sugars N-aceylgalactosamine,
galactose, or xylose to a hydroxyamino acid, most commonly serine
or threonine, although 5-hydroxyproline or 5-hydroxylysine may also
be used. Removal of glycosylation sites form an antibody is
conveniently accomplished by altering the amino acid sequence such
that one of the above-described tripeptide sequences (for N-linked
glycosylation sites) is removed. The alteration may be made by
substitution of an asparagine, serine or threonine residue within
the glycosylation site another amino acid residue (e.g., glycine,
alanine or a conservative substitution).
[0265] In any of the embodiments herein, the isolated anti-PDL1
antibody can bind to a human PDL1, for example a human PDL1 as
shown in UniProtKB/Swiss-Prot Accession No. Q9NZQ7.1, or a variant
thereof.
[0266] In some embodiments, the anti-PDL1 antibody or antigen
binding fragment thereof administered to the individual is a
composition comprising one or more pharmaceutically acceptable
carrier. Any of the pharmaceutically acceptable carriers described
herein or known in the art may be used.
[0267] In some embodiments, the anti-PDL1 antibody described herein
is in a formulation comprising the antibody at an amount of about
60 mg/mL, histidine acetate in a concentration of about 20 mM,
sucrose in a concentration of about 120 mM, and polysorbate (e.g.,
polysorbate 20) in a concentration of 0.04% (w/v), and the
formulation has a pH of about 5.8. In some embodiments, the
anti-PDL1 antibody described herein is in a formulation comprising
the antibody in an amount of about 125 mg/mL, histidine acetate in
a concentration of about 20 mM, sucrose is in a concentration of
about 240 mM, and polysorbate (e.g., polysorbate 20) in a
concentration of 0.02% (w/v), and the formulation has a pH of about
5.5.
C. VEGF Antagonists
[0268] Certain aspects of the present disclosure relate to methods
of treating or delaying progression of cancer using an
anti-angiogenesis agent (e.g., a VEGF antagonist such as an
anti-VEGF antibody) in combination with a PD-1 axis binding
antagonist (e.g., anti-PD-L1 antibody) and an anti-OX40 antibody
(e.g., an antibody that binds human OX40).
[0269] As used herein, an "anti-angiogenesis agent" or
"angiogenesis inhibitor" refers to a small molecular weight
substance, a polynucleotide, a polypeptide, an isolated protein, a
recombinant protein, an antibody, or conjugates or fusion proteins
thereof, that inhibits angiogenesis, vasculogenesis, or undesirable
vascular permeability, either directly or indirectly. It should be
understood that the anti-angiogenesis agent includes those agents
that bind and block the angiogenic activity of the angiogenic
factor or its receptor. For example, an anti-angiogenesis agent is
an antibody or other antagonist to an angiogenic agent as defined
throughout the specification or known in the art, e.g., but are not
limited to, antibodies to VEGF-A or to the VEGF-A receptor (e.g.,
KDR receptor or Flt-1 receptor), VEGF-trap, anti-PDGFR inhibitors
such as Gleevec.TM. (Imatinib Mesylate). Anti-angiogenesis agents
also include native angiogenesis inhibitors, e.g., angiostatin,
endostatin, etc. See, e.g., Klagsbrun and D'Amore, Annu. Rev.
Physiol., 53:217-39 (1991); Streit and Detmar, Oncogene,
22:3172-3179 (2003) (e.g., Table 3 listing anti-angiogenic therapy
in malignant melanoma); Ferrara & Alitalo, Nature Medicine
5:1359-1364 (1999); Tonini et al., Oncogene, 22:6549-6556 (2003)
(e.g., Table 2 listing known antiangiogenic factors); and Sato.
Int. J. Clin. Oncol., 8:200-206 (2003) (e.g., Table 1 lists
anti-angiogenic agents used in clinical trials).
[0270] As used herein, the term "VEGF" or "VEGF-A" is used to refer
to the 165-amino acid human vascular endothelial cell growth factor
and related 121-, 145-, 189-, and 206-amino acid human vascular
endothelial cell growth factors, as described by, e.g., Leung et
al. Science, 246:1306 (1989), and Houck et al. Mol. Endocrin.,
5:1806 (1991), together with the naturally occurring allelic and
processed forms thereof. VEGF-A is part of a gene family including
VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and P1GF. VEGF-A primarily
binds to two high affinity receptor tyrosine kinases, VEGFR-1
(Flt-1) and VEGFR-2 (Flk-1/KDR), the latter being the major
transmitter of vascular endothelial cell mitogenic signals of
VEGF-A. Additionally, neuropilin-1 has been identified as a
receptor for heparin-binding VEGF-A isoforms, and may play a role
in vascular development. The term "VEGF" or "VEGF-A" also refers to
VEGFs from non-human species such as mouse, rat, or primate.
Sometimes the VEGF from a specific species is indicated by terms
such as hVEGF for human VEGF or mVEGF for murine VEGF. Typically,
VEGF refers to human VEGF. The term "VEGF" is also used to refer to
truncated forms or fragments of the polypeptide comprising amino
acids 8 to 109 or 1 to 109 of the 165-amino acid human vascular
endothelial cell growth factor. Reference to any such forms of VEGF
may be identified in the application, e.g., by "VEGF (8-109),"
"VEGF (1-109)" or "VEGF165." The amino acid positions for a
"truncated" native VEGF are numbered as indicated in the native
VEGF sequence. For example, amino acid position 17 (methionine) in
truncated native VEGF is also position 17 (methionine) in native
VEGF. The truncated native VEGF has binding affinity for the KDR
and Flt-1 receptors comparable to native VEGF.
[0271] As used herein, a "chimeric VEGF receptor protein" is a VEGF
receptor molecule having amino acid sequences derived from at least
two different proteins, at least one of which is a VEGF receptor
protein. In certain embodiments, the chimeric VEGF receptor protein
is capable of binding to and inhibiting the biological activity of
VEGF.
[0272] As used herein, a "VEGF antagonist" or "VEGF-specific
antagonist" refers to a molecule capable of binding to VEGF,
reducing VEGF expression levels, or neutralizing, blocking,
inhibiting, abrogating, reducing, or interfering with VEGF
biological activities, including, but not limited to, VEGF binding
to one or more VEGF receptors, VEGF signaling, and VEGF mediated
angiogenesis and endothelial cell survival or proliferation. For
example, a molecule capable of neutralizing, blocking, inhibiting,
abrogating, reducing, or interfering with VEGF biological
activities can exert its effects by binding to one or more VEGF
receptor (VEGFR) (e.g., VEGFR1, VEGFR2, VEGFR3, membrane-bound VEGF
receptor (mbVEGFR), or soluble VEGF receptor (sVEGFR)). Included as
VEGF-specific antagonists useful in the methods of the invention
are polypeptides that specifically bind to VEGF, anti-VEGF
antibodies and antigen-binding fragments thereof, receptor
molecules and derivatives which bind specifically to VEGF thereby
sequestering its binding to one or more receptors, fusions proteins
(e.g., VEGF-Trap (Regeneron)), and VEGF.sub.121-gelonin
(Peregrine). VEGF-specific, antagonists also include antagonist
variants of VEGF polypeptides, antisense nucleobase oligomers
complementary to at least a fragment of a nucleic acid molecule
encoding a VEGF polypeptide; small RNAs complementary to at least a
fragment of a nucleic acid molecule encoding a VEGF polypeptide;
ribozymes that target VEGF; peptibodies to VEGF; and VEGF aptamers.
VEGF antagonists also include polypeptides that bind to VEGFR,
anti-VEGFR antibodies, and antigen-binding fragments thereof, and
derivatives which bind to VEGFR thereby blocking, inhibiting,
abrogating, reducing, or interfering with VEGF biological
activities (e.g., VEGF signaling), or fusions proteins.
VEGF-specific antagonists also include nonpeptide small molecules
that bind to VEGF or VEGFR and are capable of blocking, inhibiting,
abrogating, reducing, or interfering with VEGF biological
activities. Thus, the term "VEGF activities" specifically includes
VEGF mediated biological activities of VEGF. In certain
embodiments, the VEGF antagonist reduces or inhibits, by at least
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, the expression
level or biological activity of VEGF. In some embodiments, the VEGF
inhibited by the VEGF-specific antagonist is VEGF (8-109). VEGF
(1-109), or VEGF.sub.165.
[0273] As used herein, VEGF antagonists can include, but are not
limited to, anti-VEGFR2 antibodies and related molecules
ramucirumab, tanibirumab, aflibercept), anti-VEGFR1 antibodies and
related molecules (e.g., icrucumab, aflibercept (VEGF Trap-Eye;
EYLEA.RTM.), and ziv-aflibercept (VEGF Trap; ZALTRAP.RTM.)),
bispecific VEGF antibodies (e.g., MP-0250, vanucizumab (VEGF-ANG2),
and bispecific antibodies disclosed in US 2001/0236388), bispecific
antibodies including combinations of two of anti-VEGF, anti-VEGFR1,
and anti-VEGFR2 arms, anti-VEGFA antibodies (e.g., bevacizumab,
sevacizumab), anti-VEGFB antibodies, anti-VEGFC antibodies (e.g.,
VGX-100), anti-VEGFD antibodies, and nonpeptide small molecule VEGF
antagonists (e.g., pazopanih, axitinib, vandetanib, stivarga,
cabozantinib, lenvatinib, nintedanib, orantinib, telatinib,
dovitinig, cediranib, motesanib, sulfatinib, apatinib, foretinib,
famitinib, and tivozanib).
[0274] As used herein, an "anti-VEGF antibody" is an antibody that
binds to VEGF with sufficient affinity and specificity. In certain
embodiments, the antibody will have a sufficiently high binding
affinity for VEGF, for example, the antibody may bind hVEGF with a
K.sub.d value of between 100 nM-1 pM. Antibody affinities may be
determined, e.g., by a surface plasmon resonance based assay (such
as the BIAcore assay as described in PCT Application Publication
No. WO2005/012359); enzyme-linked munoabsorbent assay (ELISA); and
competition assays (e.g. RIA's). In certain embodiments, the
anti-VEGF antibody can be used as a therapeutic agent in targeting
and interfering with diseases or conditions wherein the VEGF
activity is involved. Also the antibody may be subjected to other
biological activity assays, e.g., in order to evaluate its
effectiveness as a therapeutic. Such assays are known the art and
depend on the target antigen and intended use for the antibody.
Examples include the HUVEC inhibition assay; tumor cell growth
inhibition assays (as described in WO 89/06692, for example);
antibody-dependent cellular cytotoxicity (ADCC) and
complement-mediated cytotoxicity (CDC) assays (U.S. Pat. No.
5,500,362); and agonistic activity or hematopoiesis assays (see WO
95/27062). An anti-VEGF antibody will usually not bind, to other
VEGF homologues such as VEGF-B or VEGF-C, nor other growth factors
such as PIGF, PDGF, or bFGF. In one embodiment, anti-VEGF antibody
is a monoclonal antibody that binds to the same epitope as the
monoclonal anti-VEGF antibody A4.6.1 produced by hybridoma ATCC HB
10709. In another embodiment, the anti-VEGF antibody is a
recombinant humanized anti-VEGF monoclonal antibody generated
according to Presta et al. (1997) Cancer Res. 57:4593-4599,
including but not limited to the antibody known as bevacizumab (BV;
AVASTIN.RTM.).
[0275] As used herein, the anti-VEGF antibody "Bevacizumab (BY),"
also known as "rhuMAb VEGF" or "AVASTIN.RTM.," is a recombinant
humanized anti-VEGF monoclonal antibody generated according to
Presta et al. (1997) Cancer Res. 57:4593-4599. It comprises mutated
human IgG1 framework regions and antigen-binding
complementarity-determining regions from the murine anti-hVEGF
monoclonal antibody A.4.6.1 that blocks binding of human VEGF to
its receptors.
[0276] Approximately 93% of the amino acid sequence of bevacizumab,
including most of the framework regions, is derived from human
IgG1, and about 7% of the sequence is derived from the murine
antibody A4.6.1. Bevacizumab has a molecular mass of about 149,000
daltons and is glycosylated. Bevacizumab and other humanized
anti-VEGF antibodies are further described in U.S. Pat. No.
6,884,879 issued Feb. 26, 2005, the entire disclosure of which is
expressly incorporated herein by reference. Additional preferred
antibodies include the G6 or B20 series antibodies (e.g., G6-31,
B20-4.1), as described in PCT Application Publication No. WO
2005/012359. For additional preferred antibodies see U.S. Pat. Nos.
7,060,269, 6,582,959, 6,703,020; 6,054,297; WO98/45332; WO
96/30046; WO94/10202; EP 0666868B1; U.S. Patent Application
Publication Nos. 2006009360, 20050186208, 20030206899, 20030190317,
20030203409, and 20050112126; and Popkov et al., Journal of
Immunological Methods 288:149-164 (2004). Other preferred
antibodies include those that bind to a functional epitope on human
VEGF comprising of residues F17, M18, D19, Y:21, Y25, Q89, 191,
K101, E103, and C104 or, alternatively, comprising residues F17,
Y21, Q22, Y25, D63, 183, and Q89.
[0277] As used herein, the "epitope A4.6.1" refers to the epitope
recognized by the anti-VEGF antibody bevacizumab (AVASTIN.RTM.)
(see Muller Y et al., Structure 15 Sep. 1998, 6:1153-1167). In
certain embodiments of the invention, the anti-VEGF antibodies
include, but are not limited to, a monoclonal antibody that binds
to the same epitope as the monoclonal anti-VEGF antibody A4.6.1
produced by hybridoma ATCC HB 10709; a recombinant humanized
anti-VEGF monoclonal antibody generated according to Presta et al.
(1997) Cancer Res. 57:4593-4599.
[0278] As described supra, an anti-angiogenesis agent may include a
compound such as a small molecular weight substance, a
polynucleotide, a polypeptide, an isolated protein, a recombinant
protein, an antibody, or conjugates or fusion proteins thereof. In
some embodiments, the anti-angiogenesis agent is an anti-VEGFR2
antibody; an anti-VEGFR1 antibody; a VEGF-trap; a bispecific VEGF
antibody; a bispecific antibody comprising a combination of two
arms selected from an anti-VEGF arm, an anti-VEGFR1 arm, and an
anti-VEGFR2 arm; an anti-VEGF-A antibody (e.g., an anti-KDR
receptor or anti-Flt-1 receptor antibody); an anti-VEGFB antibody;
an anti-VEGFC antibody; an anti-VEGFD antibody; a nonpeptide small
molecule VEGF antagonist; an anti-PDGFR inhibitor; or a native
angiogenesis inhibitor. In certain embodiments, the
anti-angiogenesis agent is ramucirumab, tanibirumab, aflibercept
(e.g., VEGF Trap-Eye; EYLEA.RTM.), icrucumab, ziv-aflibercept
(e.g., VEGF Trap; ZALTRAP.RTM.), MP-0250, vanucizumab, sevacizumab,
VGX-100, pazopanib, axitinib, vandetanib, stivarga, cabozantinib,
lenvatinib, nintedanib, orantinib, telatinib, dovitinig, cediranib,
motesanib, sulfatinib, apatinib, foretinib, famitinib, imatinib
(e.g., Imatinib Mesylate; Gleevec.TM.), and tivozanib.
[0279] In some embodiments, the anti-angiogenesis agent is an
anti-angiogenesis antibody. Descriptions of antibodies and methods
for generating antibodies are further provided infra. In some
embodiments, the anti-angiogenesis antibody is a monoclonal
antibody. In some embodiments, the anti-angiogenesis antibody is a
human or humanized antibody (described in more detail below).
[0280] In some embodiments, the anti-angiogenesis agent is a VEGF
antagonist. For example, VEGF antagonists of the present disclosure
may include without limitation polypeptides that specifically bind
to VEGF, anti-VEGF antibodies and antigen-binding fragments
thereof; receptor molecules and derivatives which bind specifically
to VEGF, thereby sequestering its binding to one or more receptors;
fusion proteins (e.g., VEGF-Trap (Regeneron)), VEGF.sub.121-gelonin
(Peregrine), antagonist variants of VEGF polypeptides, antisense
nucleobase oligormers complementary to at least a fragment of a
nucleic acid molecule encoding a VEGF polypeptide; small RNAs
complementary to at least a fragment of a nucleic acid molecule
encoding a VEGF polypeptide (e.g., an RNAi, siRNA, shRNA, or
miRNA); ribozymes that target VEGF; peptibodies to VEGF; VEGF
aptamers; polypeptides that bind to VEGFR; anti-VEGFR antibodies
and antigen-binding fragments thereof; derivatives which bind to
VEGFR thereby blocking, inhibiting, abrogating, reducing, or
interfering with VEGF biological activities (e.g., VEGF signaling);
fusion proteins; and nonpeptide small molecules that bind to VEGF
or VEGFR and are capable of blocking, inhibiting, abrogating,
reducing, or interfering with VEGF biological activities.
[0281] In certain embodiments, the VEGF antagonist reduces or
inhibits, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%
or more, the expression level or biological activity of VEGF. For
example, in some embodiments, the VEGF antagonist may reduce or
inhibit the expression level or biological activity of VEGF by at
least 10%, at least 15%, at least 20%, at least 25%, at least 30%,
at least 35%, at least 40%, at least 45%, at least 50%, at least
55%, at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, or at least 95%. In some
embodiments, the VEGF inhibited by the VEGF-specific antagonist is
VEGF (8-109), VEGF (1-109), or VEGF.sub.155.
[0282] Certain aspects of the methods, uses, and kits of the
present disclosure are based, at least in part, on the surprising
discovery that anti-VEGF treatment can improve the functional
phenotype of tumoral dendritic cells (e.g., by leading to increased
expression of MHC Class II and/or OX40L). Without wishing to be
bound to theory, this property, inter alia, may make combination
therapies including an anti-angiogenesis agent and an OX40 binding
agonist particularly advantageous for the treatment of cancer,
e.g., by resulting in enhanced anti-tumor responses such as
anti-tumoral T cell responses.
[0283] Therefore, in some embodiments, the VEGF antagonist
increases MHC class II expression on intratumoral dendritic cells,
e.g., as compared to MHC class II expression on dendritic cells
from a tumor treated with a control antibody (e.g., an isotype
control). MHC class II is known as a family of related molecules
(typically heterodimers containing alpha and beta chains) that
present antigen to T cells. As used herein, MHC class II expression
may refer to expression of any MHC class II molecule or chain,
including without limitation a polypeptide encoded by the human
genes HLA-DM alpha (e.g., NCBI Gene ID No. 3108), HLA-DM beta
(e.g., NCBI Gene ID No. 3109), HLA-DO alpha (e.g., NCBI Gene ID No.
3111), HLA-DO beta (e.g., NCBI Gene ID No. 3112), HLA-DP alpha 1
(e.g., NCBI Gene ID No. 3113), HLA-DP beta 1 (e.g., NCBI Gene ID
No. 3115), HLA-DQ alpha 1 (e.g., NCBI Gene ID No. 3117), HLA-DQ
alpha 2 (e.g., NCBI Gene ID No. 3118), HLA-DQ beta 1 (e.g., NCBI
Gene ID No. 3119), HLA-DQ beta 2 (e.g., NCBI Gene ID No. 3120),
HLA-DR alpha (e.g., NCBI Gene ID No. 3122), HLA-DR beta 1 (e.g.,
NCBI Gene ID No. 3123), HLA-DR beta 3 (e.g., NCBI Gene ID No.
3125), HLA-DR beta 4 (e.g., NCBI Gene ID No. 3126), or HLA-DR beta
5 (e.g., NCBI Gene ID No. 3127). It will be appreciated by one of
skill in the art that MHC genes are highly variable across
populations, and thus the specific genes and sequences listed are
merely exemplary and in no way intended to be limiting.
[0284] In some embodiments, the VEGF antagonist increases OX40L
expression on intratumoral dendritic cells, e.g., as compared to
OX40L expression on dendritic cells from a tumor treated with a
control antibody (e.g., an isotype control). OX40L (also known as
tumor necrosis factor ligand superfamily member 4 or CD252) is
known as the binding partner or ligand of OX40. Examples of OX40L
polypeptides including without limitation polypeptides having the
amino acid sequence represented by UniProt Accession No. P43488
and/or a polypeptide encoded by gene TNFSF4 (e.g., NCBI Gene ID No.
7292).
[0285] Methods for measuring MHC class II or OX40L expression are
known in the art and may include without limitation FACS, Western
blot, ELISA, immunoprecipitation, immunohistochemistry,
immunofluorescence, radioimmunoassay, dot blotting, immunodetection
methods, HPLC, surface plasmon resonance, optical spectroscopy,
mass spectrometery, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR,
RNA-seq, microarray analysis, SAGE, MassARRAY technique, and FISH,
and combinations thereof.
[0286] In some embodiments, the dendritic cells are myeloid
dendritic cells. In other embodiments, the dendritic cells are
non-myeloid dendritic cells (e.g., lymphoid or plasmacytoid
dendritic cells). The cell-surface antigens expressed by dendritic
cells, and those that distinguish myeloid and non-myeloid dendritic
cells, are known in the art. For example, dendritic cells may be
identified by expression of CD45, CD11c, and MHC class II. They may
be distinguished from other cell types (e.g., macrophages,
neutrophils, and granulocytic myeloid cells) by their lack of
significant F4/80 and Gr1 expression. In some embodiments, myeloid
dendritic cells are dendritic cells that express CD11b, and
non-myeloid dendritic cells are dendritic cells that lack
significant CD11 b expression. For further descriptions of myeloid
and non-myeloid dendritic cells, see, e.g., Steinman, R. M. and
Inaba, K. (1999) J. Leukoc. Biol. 66:205-8.
[0287] VEGF Receptor Molecules
[0288] In some embodiments, the anti-angiogenesis agent is a VEGF
antagonist. In some embodiments, the VEGF antagonist comprises a
soluble VEGF receptor or a soluble VEGF receptor fragment that
specifically binds to VEGF. The two best characterized VEGF
receptors are VEGFR1 (also known as Flt-1) and VEGFR2 (also known
as KDR and FLK-1 for the murine homolog). The specificity of each
receptor for each VEGF family member varies but VEGF-A binds to
both Flt-1 and KDR. Both Flt-I and KDR belong to the family of
receptor tyrosine kinases (RTKs). The RTKs comprise a large family
of transmembrane receptors with diverse biological activities. At
least nineteen (19) distinct RTK subfamilies have been identified.
The receptor tyrosine kinase (RTK) family includes receptors that
are crucial for the growth and differentiation of a variety of cell
types (Yarden and Ullrich (1988) Ann. Rev. Biochem. 57:433-478;
Ullrich and Schlessinger (1990) Cell 61:243-254). The intrinsic
function of RTKs is activated upon ligand binding, which results in
phosphorylation of the receptor and multiple cellular substrates,
and subsequently in a variety of cellular responses (Ullrich &
Schlessinger (1990) Cell 61:203-212). Thus, receptor tyrosine
kinase mediated signal transduction is initiated by extracellular
interaction with a specific growth factor (ligand), typically
followed by receptor dimerization, stimulation of the intrinsic
protein tyrosine kinase activity and receptor
trans-phosphorylation. Binding sites are thereby created for
intracellular signal transduction molecules and lead to the
formation of complexes with a spectrum of cytoplasmic signaling
molecules that facilitate the appropriate cellular response. (e.g.,
cell division, differentiation, metabolic effects, changes in the
extracellular microenvironment) see, Schlessinger and Ullrich
(1992) Neuron 9:1-20. Structurally, both Flt-1 and KDR have seven
immunoglobulin-like domains in the extracellular domain, a single
transmembrane region, and a consensus tyrosine kinase sequence
which is interrupted by a kinase-insert domain Matthews et al.
(1991) PNAS USA 88:9026-9030; Terman et al. (1991) Oncogene
6:1677-1683. The extracellular domain is involved in the binding of
VEGF and the intracellular domain is involved in signal
transduction.
[0289] VEGF receptor molecules, or fragments thereof, that
specifically bind to VEGF can be used in the methods of the
invention to bind to and sequester the VEGF protein, thereby
preventing it from signaling. In certain embodiments, the VEGF
receptor molecule, or VEGF binding fragment thereof, is a soluble
form, such as sFlt-1. A soluble form of the receptor exerts an
inhibitory effect on the biological activity of the VEGF protein by
binding to VEGF, thereby preventing it from binding to its natural
receptors present on the surface of target cells. Also included are
VEGF receptor fusion proteins, examples of which are described
below.
[0290] In some embodiments, the VEGF antagonist is a chimeric VEGF
receptor protein. A chimeric VEGF receptor protein is a receptor
molecule having amino acid sequences derived from at least two
different proteins, at least one of which is a VEGF receptor
protein (e.g., the flt-1 or KDR receptor), that is capable of
binding to and inhibiting the biological activity of VEGF. In
certain embodiments, the chimeric VEGF receptor proteins of the
invention consist of amino acid sequences derived from only two
different VEGF receptor molecules; however, amino acid sequences
comprising one, two, three, four, five, six, or all seven Ig-like
domains from the extracellular ligand-binding region of the flt-1
and/or KDR receptor can be linked to amino acid sequences from
other unrelated proteins, for example, immunoglobulin sequences.
Other amino acid sequences to which Ig-like domains are combined
will be readily apparent to those of ordinary skill in the art.
Examples of chimeric VEGF receptor proteins include, e.g., soluble
Flt-1/Fc, KDR/Fc, or FLt-1/KDR/Fc (also known as VEGF Trap). (See
for example PCT Application Publication No. WO97/44453).
[0291] A soluble VEGF receptor protein or chimeric VEGF receptor
proteins of the invention includes VEGF receptor proteins which are
not fixed to the surface of cells via a transmembrane domain. As
such, soluble forms of the VEGF receptor, including chimeric
receptor proteins, while capable of binding to and inactivating
VEGF, do not comprise a transmembrane domain and thus generally do
not become associated with the cell membrane of cells in which the
molecule is expressed.
[0292] In some embodiments, the VEGF antagonist (I, an anti-VEGF
antibody, such as bevacizumab) is administered by gene therapy.
See, for example, WO 96/0'7321 published Mar. 14, 1996 concerning
the use of gene therapy to generate intracellular antibodies. There
are two major approaches to getting the nucleic acid (optionally
contained in a vector) into the patient's cells; in vivo and ex
vivo. For in vivo delivery the nucleic acid is injected directly
into the patient, usually at the site where the antibody is
required. For ex vivo treatment, the patient's cells are removed,
the nucleic acid is introduced into these isolated cells and the
modified cells are administered to the patient either directly or,
for example, encapsulated within porous membranes which are
implanted into the patient (see, e.g. U.S. Pat. Nos. 4,892,538 and
5,283,187). There are a variety of techniques available for
introducing nucleic acids into viable cells. The techniques vary
depending upon whether the nucleic acid is transferred into
cultured cells in vitro, or in vivo in the cells of the intended
host. Techniques suitable for the transfer of nucleic acid into
mammalian cells in vitro include the use of liposomes,
electroporation, microinjection, cell fusion, DEAE-dextran, the
calcium phosphate precipitation method, etc, A commonly used vector
for ex vivo delivery of the gene is a retrovirus. The currently
preferred in vivo nucleic acid transfer techniques include
transfection with viral vectors (such as adenovirus, Herpes simplex
I virus, or adeno-associated virus) and lipid-based systems (useful
lipids for lipid-mediated transfer of the gene are DOTMA, DOPE and
DC-Cho), for example). In some situations it is desirable to
provide the nucleic acid source with an agent that targets the
target cells, such as an antibody specific for a cell surface
membrane protein or the target cell, a ligand for a receptor on the
target cell, etc. Where liposomes are employed, proteins which bind
to a cell surface membrane protein associated with endocytosis may
be used for targeting and/or to facilitate uptake, e.g. capsid
proteins or fragments thereof tropic for a particular cell type,
antibodies for proteins which undergo internalization in cycling,
and proteins that target intracellular localization and enhance
intracellular half-life. The technique of receptor-mediated
endocytosis is described, for example, by Wu et al., J. Biol. Chem.
262:44294432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA
87; 3410-3414 (1990). For review of the currently known gene
marking and gene therapy protocols see Anderson et al., Science
256:808-813 (1992), See also WO 93/25673 and the references cited
therein.
[0293] Anti-VEGF Antibodies
[0294] In some embodiments, the anti-angiogenesis agent is a VEGF
antagonist. In some embodiments, the VEGF antagonist is an
anti-VEGF antibody. In some embodiments, the anti-VEGF antibody may
be a human or humanized antibody. In some embodiments, the
anti-VEGF antibody may be a monoclonal antibody.
[0295] The VEGF antigen to be used for production of VEGF
antibodies may be, e.g., the VEGF.sub.165 molecule as well as other
isoforms of VEGF or a fragment thereof containing the desired
epitope. In one embodiment, the desired epitope is the one
recognized by bevacizumab, which binds to the same epitope as the
monoclonal anti-VEGF antibody A4.6.1 produced by hybridoma ATCC HB
10709 (known as "epitope A.4.6.1" defined herein). Other forms of
VEGF useful for generating anti-VEGF antibodies of the invention
will be apparent to those skilled in the art.
[0296] Human VEGF was obtained by first screening a cDNA library
prepared from human cells, using bovine VEGF cDNA as a
hybridization probe. Leung et al. (1989) Science, 246:1306. One
cDNA identified thereby encodes a 165-amino acid protein having
greater than 95% homology to bovine VEGF; this 165-amino acid
protein is typically referred to as human VEGF (hVEGF) or
VEGF.sub.165. The mitogenic activity of human VEGF was confirmed by
expressing the human VEGF cDNA in mammalian host cells. Media
conditioned by cells transfected with the human VEGF cDNA promoted
the proliferation of capillary endothelial cells, whereas control
cells did not. Leung et al. (1989) Science, supra. Further efforts
were undertaken to clone and express VEGF via recombinant DNA
techniques. (See, e.g., Ferrara, Laboratory Investigation
72:615-618 (1995), and the references cited therein).
[0297] VEGF is expressed in a variety of tissues as multiple
homodimeric forms (121, 145, 165, 189, and 206 amino acids per
monomer) resulting from alternative RNA splicing. VEGF.sub.121 is a
soluble mitogen that does not bind heparin; the longer forms of
VEGF bind heparin with progressively higher affinity. The
heparin-binding forms of VEGF can be cleaved in the carboxy
terminus by plasmin to release a diffusible form(s) of VEGF Amino
acid sequencing of the carboxy terminal peptide identified after
plasmin cleavage is Arg.sub.110-Ala.sub.111. Amino terminal "core"
protein, VEGF (1-110) isolated as a homodimer, binds neutralizing
monoclonal antibodies (such as the antibodies referred to as 4.6.1
and 3.2E3.1.1) and soluble forms of VEGF receptors with similar
affinity compared to the intact VEGF.sub.165 homodimer.
[0298] Several molecules structurally related to VEGF have also
been identified recently, including placenta growth factor (PIGF),
VEGF-B, VEGF-C, VEGF-D and VEGF-E. Ferrara and Davis-Smyth (1987)
Endocr. Rev., supra; Ogawa et al. J. Biological Chem.
273:31273-31281 (1998); Meyer et al. EMBO J., 18:363-374 (1999). A
receptor tyrosine kinase, Flt-4 (VEGFR-3), has been identified as
the receptor for VEGF-C and VEGF-D. Joukov et al. EMBO. J. 15:1751
(1996); Lee et al. PNAS USA 93:1988-1992 (1996); Achen et al.
(1998) PNAS USA 95:548-553. VEGF-C has been shown to be involved in
the regulation of lymphatic angiogenesis. Jeltsch et al. Science
276:1423-1425 (1997).
[0299] Two VEGF receptors have been identified, Flt-1 (also called
VEGFR-1) and KDR (also called VEGFR-2). Shibuya et al. (1990)
Oncogene 8:519-527; de Vries et al. (1992) Science 255:989-991;
Terman et al. (1992) Biochem. Biophys. Res. Commun 187:1579-1586.
Neuropilin-1 has been shown to be a selective VEGF receptor, able
to bind the heparin-binding VEGF isoforms (Soker et al. (1998) Cell
92:735-45).
[0300] Anti-VEGF antibodies that are useful in the methods of the
invention include any antibody, or antigen binding fragment
thereof, that bind with sufficient affinity and specificity to VEGF
and can reduce or inhibit the biological activity of VEGF. An
anti-VEGF antibody will usually not bind to other VEGF homologues
such as VEGF-B or VEGF-C, nor other growth factors such as P1GF,
PDGF, or bFGF.
[0301] In certain embodiments of the invention, the anti-VEGF
antibodies include, but are not limited to, a monoclonal antibody
that binds to the same epitope as the monoclonal anti-VEGF antibody
A4.6.1 produced by hybridoma ATCC HB 10709; a recombinant humanized
anti-VEGF monoclonal antibody generated according to Presta et al.
(1997) Cancer Res. 57:4593-4599. In one embodiment, the anti-VEGF
antibody is "bevacizumab (BV)", also known as "rhuMAb VEGF" or
"AVASTIN.RTM.". It comprises mutated human IgG1 framework regions
and antigen-binding complementarity-determining regions from the
murine anti-hVEGF monoclonal antibody A.4.6.1 that blocks binding
of human VEGF to its receptors. Approximately 93% of the amino acid
sequence of bevacizumab, including most of the framework regions,
is derived from human IgG1, and about 7% of the sequence is derived
from the murine antibody A4.6.1.
[0302] Bevacizumab (AVASTIN.RTM.) was the first anti-angiogenesis
therapy approved by the FDA and is approved for the treatment
metastatic colorectal cancer (first- and second-line treatment in
combination with intravenous 5-FU-based chemotherapy), advanced
non-squamous, non-small cell lung cancer (NSCLC) (first-line
treatment of unresectable, locally advanced, recurrent or
metastatic NSCLC in combination with carboplatin and paclitaxel)
and metastatic HER2-negative breast cancer (previously untreated,
metastatic HER2-negative breast cancer in combination with
paclitaxel).
[0303] Bevacizumab and other humanized anti-VEGF antibodies are
further described in U.S. Pat. No. 6,884,879 issued Feb. 26, 2005.
Additional antibodies include the G6 or B20 series antibodies
(e.g., G6-31, B20-4.1), as described in PCT Publication No.
WO2005/012359, PCT Publication No. WO2005/044853, and U.S. Patent
Application 60/991,302, the content of these patent applications
are expressly incorporated herein by reference. For additional
antibodies see U.S. Pat. Nos. 7,060,269, 6,582,959, 6,703,020;
6,054,297; WO98/45332; WO 96/30046; WO94/10202; EP 0666868B1; U.S.
Patent Application Publication Nos. 2006009360, 20050186208,
20030206899, 20030190317, 20030203409, and 20050112126; and Popkov
et al., Journal of Immunological Methods 288:149-164 (2004). Other
antibodies include those that bind to a functional epitope on human
VEGF comprising of residues F17, M18, D19, Y21, Y25, Q89, 1191,
K101, E103, and C104 or, alternatively, comprising residues F17,
Y21, Q22, Y25, D63, 183 and Q89.
[0304] In one embodiment of the invention, the anti-VEGF antibody
has a light chain variable region comprising the following amino
acid sequence:
TABLE-US-00016 (SEQ ID NO: 214) DIQMTQSPSS LSASVGDRVT ITCSASQDIS
NYLNWYQQKP GKAPKVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ
YSTVPWTFGQ GTKVEIKR.;
and/or a heavy chain variable region comprising the following amino
acid sequence: EVQLVESGGG LVQPGGSLRL SCAASGYTFT NYGMNWVRQA
PGKGLEWVGW INTYTGEPTY AADFKRRFTF SLDTSKSTAY LQMNSLRAED TAVYYCAKYP
HYYGSSHWYF DVWGQGTLVT VSS (SEQ ID NO:215).
[0305] In some embodiments, the anti-VEGF antibody comprises one,
two, three, four, five, or six hypervariable region (HVR) sequences
of bevacizumab. In some embodiments, the anti-VEGF antibody
comprises one, two, three, four, five, or six hypervariable region
(HVR) sequences of selected from (a) HVR-H1 comprising the amino
acid sequence of GYTFTNYGMN (SEQ ID NO:216); (b) HVR-H2 comprising
the amino acid sequence of WINTYTGEPTYAADFKR (SEQ ID NO:217); (c)
HVR-H3 comprising the amino acid sequence of YPHYYGSSHWYFDV (SEQ ID
NO:218); (d) HVR-L1 comprising the amino acid sequence of
SASQDISNYLN (SEQ ID NO:219); (e) HVR-L2 comprising the amino acid
sequence of FTSSLHS (SEQ ID NO:220); and (f) HVR-L3 comprising the
amino acid sequence of QQYSTVPWT (SEQ ID NO:221). In some
embodiments, the anti-VEGF antibody comprises one, two, three,
four, five, or six hypervariable region (HVR) sequences of an
antibody described in U.S. Pat. No. 6,884,879. In some embodiments,
the anti-VEGF antibody comprises one, two, or three hypervariable
region (HVR) sequences of a light chain variable region comprising
the following amino acid sequence: DIQMTQSPSS LSASVGDRVT ITCSASQDIS
NYLNWYQQKP GKAPKVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ
YSTVPWTFGQ GTKVEIKR. (SEQ ID NO:214) and/or one, two, or three
hypervariable region (HVR) sequences of a heavy chain variable
region comprising the following amino acid sequence: EVQLVESGGG
LVQPGGSLRL SCAASGYTFT NYGMNWVRQA PGKGLEWVGW INTYTGEPTY AADFKRRFTF
SLDTSKSTAY LQMNSLRAED TAVYYCAKYP HYYGSSHWYF DVWGQGTLVT VSS (SEQ ID
NO:215).
[0306] A "G6 series antibody" according to this invention, is an
anti-VEGF antibody that is derived from a sequence of a G6 antibody
or G6-derived antibody according to any one of FIGS. 7, 24-26, and
34-35 of PCT Publication No. WO2005/012359, the entire disclosure
of which is expressly incorporated herein by reference. See also
PCT Publication No. WO2005/044853, the entire disclosure of which
is expressly incorporated herein by reference. In one embodiment,
the G6 series antibody binds to a functional epitope on human VEGF
comprising residues F17, Y21, Q22, Y25, D63, 183 and Q89.
[0307] A "B20 series antibody" according to this invention is an
anti-VEGF antibody that is derived from a sequence of the B20
antibody or a B20-derived antibody according to any one of FIGS.
27-29 of PCT Publication No. WO2005/012359, the entire disclosure
of which is expressly incorporated herein by reference. See also
PCT Publication No. WO2005/044853, and U.S. Patent Application
60/991,302, the content of these patent applications are expressly
incorporated herein by reference. In one embodiment, the B20 series
antibody binds to a functional epitope on human VEGF comprising
residues F17, M18, D19, Y21, Y25, Q89, 191, K101, E103, and
C104.
[0308] A "functional epitope" according to this invention refers to
amino acid residues of an antigen that contribute energetically to
the binding of an antibody. Mutation of any one of the
energetically contributing residues of the antigen (for example,
mutation of wild-type VEGF by alanine or homolog mutation) will
disrupt the binding of the antibody such that the relative affinity
ratio (IC50mutant VEGF/IC50wild-type VEGF) of the antibody will be
greater than 5 (see Example 2 of WO2005/012359). In one embodiment,
the relative affinity ratio is determined by a solution binding
phage displaying ELISA. Briefly, 96-well Maxisorp immunoplates
(NUNC) are coated overnight at 4.degree. C. with an Fab form of the
antibody to be tested at a concentration of 2 .mu.g/ml in PBS, and
blocked with PBS, 0.5% BSA, and 0.05% Tween20 (PBT) for 2 h at room
temperature. Serial dilutions of phage displaying hVEGF alanine
point mutants (residues 8-109 form) or wild type hVEGF (8-109) in
PBT are first incubated on the Fab-coated plates for 15 min at room
temperature, and the plates are washed with PBS, 0.05% Tween20
(PBST). The bound phage is detected with an anti-M13 monoclonal
antibody horseradish peroxidase (Amersham Pharmacia) conjugate
diluted 1:5000 in PBT, developed with
3,3',5,5'-tetramethylbenzidine (TMB, Kirkegaard & Perry Labs,
Gaithersburg, Md.) substrate for approximately 5 min, quenched with
1.0 M H3PO4, and read spectrophotometrically at 450 nm. The ratio
of IC50 values (IC50, ala/IC50, wt) represents the fold of
reduction in binding affinity (the relative binding affinity).
[0309] 1. Antibody Affinity
[0310] In a further aspect, an anti-OX40 antibody, anti-PDL1
antibody, or anti-VEGF antibody according to any of the above
embodiments may incorporate any of the features, singly or in
combination, as described in Sections 1-7 below:
[0311] In certain embodiments, an antibody provided herein has a
dissociation constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.100 nM,
.ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM, .ltoreq.0.01 nM, or
.ltoreq.0.001 nM (e.g. 10.sup.-8 M or less, e.g. from 10.sup.-8 M
to 10.sup.-13 M, e.g., from 10.sup.-9 M to 10.sup.-13 M).
[0312] In one embodiment, Kd is measured by a radiolabeled antigen
binding assay (RIA). In one embodiment, an RIA is performed with
the Fab version of an antibody of interest and its antigen. For
example, solution binding affinity of Fabs for antigen is measured
by equilibrating Fab with a minimal concentration of
(.sup.125I)-labeled antigen in the presence of a titration series
of unlabeled antigen, then capturing bound antigen with an anti-Fab
antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.
293:865-881(1999)). To establish conditions for the assay,
MICROTITER.RTM. multi-well plates (Thermo Scientific) are coated
overnight with 5 .mu.g/ml of a capturing anti-Fab antibody (Cappel
Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked
with 2% (w/v) bovine serum albumin in PBS for two to five hours at
room temperature (approximately 23.degree. C.). In a non-adsorbent
plate (Nunc #269620), 100 pM or 26 pM [125I]-antigen are mixed with
serial dilutions of a Fab of interest (e.g., consistent with
assessment of the anti-VEGF antibody, Fab-12, in Presta et al.,
Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then
incubated overnight; however, the incubation may continue for a
longer period (e.g., about 65 hours) to ensure that equilibrium is
reached. Thereafter, the mixtures are transferred to the capture
plate for incubation at room temperature (e.g., for one hour). The
solution is then removed and the plate washed eight times with 0.1%
polysorbate 20 (TWEEN-20.RTM.) in PBS. When the plates have dried,
150 .mu.l/well of scintillant (MICROSCINT-20.TM.; Packard) is
added, and the plates are counted on a TOPCOUNT.TM. gamma counter
(Packard) for ten minutes. Concentrations of each Fab that give
less than or equal to 20% of maximal binding are chosen for use in
competitive binding assays.
[0313] According to another embodiment, Kd is measured using a
BIACORE.RTM. surface plasmon resonance assay. For example, an assay
using a BIACORE.RTM.-2000 or a BIACORE.RTM.-3000 (BIAcore, Inc.,
Piscataway, N.J.) is performed at 25.degree. C. with immobilized
antigen CM5 chips at -10 response units (RU). In one embodiment,
carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) are
activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide
hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the
supplier's instructions. Antigen is diluted with 10 mM sodium
acetate, pH 4.8, to 5 .mu.g/ml (.about.0.2 .mu.M) before injection
at a flow rate of 5 .mu.l/minute to achieve approximately 10
response units (RU) of coupled protein. Following the injection of
antigen, 1 M ethanolamine is injected to block unreacted groups.
For kinetics measurements, two-fold serial dilutions of Fab (0.78
nM to 500 nM) are injected in PBS with 0.05% polysorbate 20
(TWEEN-20.TM.) surfactant (PBST) at 25.degree. C. at a flow rate of
approximately 25 .mu.l/min Association rates (k.sub.on) and
dissociation rates (k.sub.off) are calculated using a simple
one-to-one Langmuir binding model (BIACORE.RTM. Evaluation Software
version 3.2) by simultaneously fitting the association and
dissociation sensorgrams. The equilibrium dissociation constant
(Kd) is calculated as the ratio k.sub.off/k.sub.on. See, e.g., Chen
et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds
106 M-1 s-1 by the surface plasmon resonance assay above, then the
on-rate can be determined by using a fluorescent quenching
technique that measures the increase or decrease in fluorescence
emission intensity (excitation=295 nm; emission=340 nm, 16 nm
band-pass) at 25.degree. C. of a 20 nM anti-antigen antibody (Fab
form) in PBS, pH 7.2, in the presence of increasing concentrations
of antigen as measured in a spectrometer, such as a stop-flow
equipped spectrophometer (Aviv Instruments) or a 8000-series
SLM-AMINCO.TM. spectrophotometer (ThermoSpectronic) with a stirred
cuvette.
[0314] 2. Antibody Fragments
[0315] In certain embodiments, an antibody provided herein is an
antibody fragment. Antibody fragments include, but are not limited
to, Fab, Fab', Fab'-SH, F(ab').sub.2, Fv, and scFv fragments, and
other fragments described below. For a review of certain antibody
fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a
review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology
of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,
(Springer-Verlag, New York), pp. 269-315 (1994); see also WO
93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For
discussion of Fab and F(ab').sub.2 fragments comprising salvage
receptor binding epitope residues and having increased in vivo
half-life, see U.S. Pat. No. 5,869,046.
[0316] Diabodies are antibody fragments with two antigen-binding
sites that may be bivalent or bispecific. See, for example, EP
404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003);
and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448
(1993). Triabodies and tetrabodies are also described in Hudson et
al., Nat. Med. 9:129-134 (2003).
[0317] Single-domain antibodies are antibody fragments comprising
all or a portion of the heavy chain variable domain or all or a
portion of the light chain variable domain of an antibody. In
certain embodiments, a single-domain antibody is a human
single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g.,
U.S. Pat. No. 6,248,516 B1).
[0318] Antibody fragments can be made by various techniques,
including but not limited to proteolytic digestion of an intact
antibody as well as production by recombinant host cells (e.g. E.
coli or phage), as described herein.
[0319] 3. Chimeric and Humanized Antibodies
[0320] In certain embodiments, an antibody provided herein is a
chimeric antibody. Certain chimeric antibodies are described, e.g.,
in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad.
Sci. USA, 81:6851-6855 (1984)). In one example, a chimeric antibody
comprises a non-human variable region (e.g., a variable region
derived from a mouse, rat, hamster, rabbit, or non-human primate,
such as a monkey) and a human constant region. In a further
example, a chimeric antibody is a "class switched" antibody in
which the class or subclass has been changed from that of the
parent antibody. Chimeric antibodies include antigen-binding
fragments thereof.
[0321] In certain embodiments, a chimeric antibody is a humanized
antibody. Typically, a non-human antibody is humanized to reduce
immunogenicity to humans, while retaining the specificity and
affinity of the parental non-human antibody. Generally, a humanized
antibody comprises one or more variable domains in which HVRs,
e.g., CDRs, (or portions thereof) are derived from a non-human
antibody, and FRs (or portions thereof) are derived from human
antibody sequences. A humanized antibody optionally will also
comprise at least a portion of a human constant region. In some
embodiments, some FR residues in a humanized antibody are
substituted with corresponding residues from a non-human antibody
(e.g., the antibody from which the HVR residues are derived), e.g.,
to restore or improve antibody specificity or affinity.
[0322] Humanized antibodies and methods of making them are
reviewed, e.g., in Almagro and Fransson, Front. Biosci.
13:1619-1633 (2008), and are further described, e.g., in Riechmann
et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad.
Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337,
7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods
36:25-34 (2005) (describing specificity determining region (SDR)
grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing
"resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005)
(describing "FR shuffling"); and Osbourn et al., Methods 36:61-68
(2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000)
(describing the "guided selection" approach to FR shuffling).
[0323] Human framework regions that may be used for humanization
include but are not limited to: framework regions selected using
the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296
(1993)); framework regions derived from the consensus sequence of
human antibodies of a particular subgroup of light or heavy chain
variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci.
USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623
(1993)); human mature (somatically mutated) framework regions or
human germline framework regions (see, e.g., Almagro and Fransson,
Front. Biosci. 13:1619-1633 (2008)); and framework regions derived
from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem.
272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.
271:22611-22618 (1996)).
[0324] 4. Human Antibodies
[0325] In certain embodiments, an antibody provided herein is a
human antibody. Human antibodies can be produced using various
techniques known in the art. Human antibodies are described
generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:
368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459
(2008).
[0326] Human antibodies may be prepared by administering an
immunogen to a transgenic animal that has been modified to produce
intact human antibodies or intact antibodies with human variable
regions in response to antigenic challenge. Such animals typically
contain all or a portion of the human immunoglobulin loci, which
replace the endogenous immunoglobulin loci, or which are present
extrachromosomally or integrated randomly into the animal's
chromosomes. In such transgenic mice, the endogenous immunoglobulin
loci have generally been inactivated. For review of methods for
obtaining human antibodies from transgenic animals, see Lonberg,
Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Pat. Nos.
6,075,181 and 6,150,584 describing XENOMOUSE.TM. technology; U.S.
Pat. No. 5,770,429 describing HUMAB.RTM. technology; U.S. Pat. No.
7,041,870 describing K-M MOUSE.RTM. technology, and U.S. Patent
Application Publication No. US 2007/0061900, describing
VELOCIMOUSE.RTM. technology). Human variable regions from intact
antibodies generated by such animals may be further modified, e.g.,
by combining with a different human constant region.
[0327] Human antibodies can also be made by hybridoma-based
methods. Human myeloma and mouse-human heteromyeloma cell lines for
the production of human monoclonal antibodies have been described.
(See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al.,
Monoclonal Antibody Production Techniques and Applications, pp.
51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J.
Immunol., 147: 86 (1991).) Human antibodies generated via human
B-cell hybridoma technology are also described in Li et al., Proc.
Natl. Acad. Sci. USA, 103:3557-3562 (2006), Additional methods
include those described, for example, in U.S. Pat. No. 7,189,826
(describing production of monoclonal human IgM antibodies from
hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268
(2006) (describing human-human hybridomas). Human hybridoma
technology (Trioma technology) is also described in Vollmers and
Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and
Vollmers and Brandlein, Methods and Findings in Experimental and
Clinical Pharmacology, 27(3):185-91 (2005).
[0328] Human antibodies may also be generated by isolating Fv clone
variable domain sequences selected from human-derived phage display
libraries. Such variable domain sequences may then be combined with
a desired human constant domain. Techniques for selecting human
antibodies from antibody libraries are described below.
[0329] 5. Library-Derived Antibodies
[0330] Antibodies of the invention may be isolated by screening
combinatorial libraries for antibodies with the desired activity or
activities. For example, a variety of methods are known in the art
for generating phage display libraries and screening such libraries
for antibodies possessing the desired binding characteristics. Such
methods are reviewed, e.g., in Hoogenboom et al. in Methods in
Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press,
Totowa, N.J., 2001) and further described, e.g., in the McCafferty
et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628
(1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and
Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed.,
Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.
338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093
(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472
(2004); and Lee et al., J. Immunol. Methods 284(1-2):
119-132(2004).
[0331] In certain phage display methods, repertoires of VH and VL
genes are separately cloned by polymerase chain reaction (PCR) and
recombined randomly in phage libraries, which can then be screened
for antigen-binding phage as described in Winter et al., Ann. Rev.
Immunol., 12: 433-455 (1994). Phage typically display antibody
fragments, either as single-chain Fv (scFv) fragments or as Fab
fragments. Libraries from immunized sources provide high-affinity
antibodies to the immunogen without the requirement of constructing
hybridomas. Alternatively, the naive repertoire can be cloned
(e.g., from human) to provide a single source of antibodies to a
wide range of non-self and also self antigens without any
immunization as described by Griffiths et al., EMBO J, 12: 725-734
(1993). Finally, naive libraries can also be made synthetically by
cloning unrearranged V-gene segments from stem cells, and using PCR
primers containing random sequence to encode the highly variable
CDR3 regions and to accomplish rearrangement in vitro, as described
by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
Patent publications describing human antibody phage libraries
include, for example: U.S. Pat. No. 5,750,373, and US Patent
Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000,
2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and
2009/0002360.
[0332] Antibodies or antibody fragments isolated from human
antibody libraries are considered human antibodies or human
antibody fragments herein.
[0333] 6. Multispecific Antibodies
[0334] In certain embodiments, an antibody provided herein is a
multispecific antibody, e.g. a bispecific antibody. Multispecific
antibodies are monoclonal antibodies that have binding
specificities for at least two different sites. In certain
embodiments, one of the binding specificities is for OX40 and the
other is for any other antigen. In certain embodiments, bispecific
antibodies may bind to two different epitopes of OX40. Bispecific
antibodies may also be used to localize cytotoxic agents to cells
which express OX40. Bispecific antibodies can be prepared as full
length antibodies or antibody fragments.
[0335] Techniques for making multispecific antibodies include, but
are not limited to, recombinant co-expression of two immunoglobulin
heavy chain-light chain pairs having different specificities (see
Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and
Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole"
engineering (see, e.g., U.S. Pat. No. 5,731,168). Multi-specific
antibodies may also be made by engineering electrostatic steering
effects for making antibody Fc-heterodimeric molecules (WO
2009/089004A1); cross-linking two or more antibodies or fragments
(see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science,
229: 81 (1985)); using leucine zippers to produce bi-specific
antibodies (see, e.g., Kostelny et al., J. Immunol.,
148(5):1547-1553 (1992)); using "diabody" technology for making
bispecific antibody fragments (see, e.g., Hollinger et al., Proc.
Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain
Fv (sFv) dimers (see,e.g. Gruber et al., J. Immunol., 152:5368
(1994)); and preparing trispecific antibodies as described, e.g.,
in Tutt et al. J. Immunol. 147: 60 (1991).
[0336] Engineered antibodies with three or more functional antigen
binding sites, including "Octopus antibodies," are also included
herein (see, e.g. US 2006/0025576A1).
[0337] The antibody or fragment herein also includes a "Dual Acting
FAb" or "DAF" comprising an antigen binding site that binds to OX40
as well as another, different antigen (see, US 2008/0069820, for
example).
[0338] 7. Antibody Variants
[0339] In certain embodiments, amino acid sequence variants of the
antibodies provided herein are contemplated. For example, it may be
desirable to improve the binding affinity and/or other biological
properties of the antibody. Amino acid sequence variants of an
antibody may be prepared by introducing appropriate modifications
into the nucleotide sequence encoding the antibody, or by peptide
synthesis. Such modifications include, for example, deletions from,
and/or insertions into and/or substitutions of residues within the
amino acid sequences of the antibody. Any combination of deletion,
insertion, and substitution can be made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics, e.g., antigen-binding.
[0340] a) Substitution, Insertion, and Deletion Variants
[0341] In certain embodiments, antibody variants having one or more
amino acid substitutions are provided. Sites of interest for
substitutional mutagenesis include the HVRs and FRs. Conservative
substitutions are shown in Table A under the heading of "preferred
substitutions." More substantial changes are provided in Table A
under the heading of "exemplary substitutions," and as further
described below in reference to amino acid side chain classes Amino
acid substitutions may be introduced into an antibody of interest
and the products screened for a desired activity, e.g.,
retained/improved antigen binding, decreased immunogenicity, or
improved ADCC or CDC.
TABLE-US-00017 TABLE A Original Exemplary Preferred Residue
Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys;
Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn
Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp
Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val;
Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met;
Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu
Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)
Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe;
Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu
[0342] Amino acids may be grouped according to common side-chain
properties:
[0343] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
[0344] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0345] (3) acidic: Asp, Glu;
[0346] (4) basic: His, Lys, Arg;
[0347] (5) residues that influence chain orientation: Gly, Pro;
[0348] (6) aromatic: Trp, Tyr, Phe.
[0349] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0350] One type of substitutional variant involves substituting one
or more hypervariable region residues of a parent antibody (e.g. a
humanized or human antibody). Generally, the resulting variant(s)
selected for further study will have modifications (e.g.,
improvements) in certain biological properties (e.g., increased
affinity, reduced immunogenicity) relative to the parent antibody
and/or will have substantially retained certain biological
properties of the parent antibody. An exemplary substitutional
variant is an affinity matured antibody, which may be conveniently
generated, e.g., using phage display-based affinity maturation
techniques such as those described herein. Briefly, one or more HVR
residues are mutated and the variant antibodies displayed on phage
and screened for a particular biological activity (e.g. binding
affinity).
[0351] Alterations (e.g., substitutions) may be made in HVRs, e.g.,
to improve antibody affinity. Such alterations may be made in HVR
"hotspots," i.e., residues encoded by codons that undergo mutation
at high frequency during the somatic maturation process (see, e.g.,
Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or residues
that contact antigen, with the resulting variant VH or VL being
tested for binding affinity. Affinity maturation by constructing
and reselecting from secondary libraries has been described, e.g.,
in Hoogenboom et al. in Methods in Molecular Biology 178:1-37
(O'Brien et al., ed., Human Press, Totowa, N.J., (2001).) In some
embodiments of affinity maturation, diversity is introduced into
the variable genes chosen for maturation by any of a variety of
methods (e.g., error-prone PCR, chain shuffling, or
oligonucleotide-directed mutagenesis). A secondary library is then
created. The library is then screened to identify any antibody
variants with the desired affinity. Another method to introduce
diversity involves HVR-directed approaches, in which several HVR
residues (e.g., 4-6 residues at a time) are randomized HVR residues
involved in antigen binding may be specifically identified, e.g.,
using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3
in particular are often targeted.
[0352] In certain embodiments, substitutions, insertions, or
deletions may occur within one or more HVRs so long as such
alterations do not substantially reduce the ability of the antibody
to bind antigen. For example, conservative alterations (e.g.,
conservative substitutions as provided herein) that do not
substantially reduce binding affinity may be made in HVRs. Such
alterations may, for example, be outside of antigen contacting
residues in the HVRs. In certain embodiments of the variant VH and
VL sequences provided above, each HVR either is unaltered, or
contains no more than one, two or three amino acid
substitutions.
[0353] A useful method for identification of residues or regions of
an antibody that may be targeted for mutagenesis is called "alanine
scanning mutagenesis" as described by Cunningham and Wells (1989)
Science, 244:1081-1085. In this method, a residue or group of
target residues (e.g., charged residues such as arg, asp, his, lys,
and glu) are identified and replaced by a neutral or negatively
charged amino acid (e.g., alanine or polyalanine) to determine
whether the interaction of the antibody with antigen is affected.
Further substitutions may be introduced at the amino acid locations
demonstrating functional sensitivity to the initial substitutions.
Alternatively, or additionally, a crystal structure of an
antigen-antibody complex to identify contact points between the
antibody and antigen. Such contact residues and neighboring
residues may be targeted or eliminated as candidates for
substitution. Variants may be screened to determine whether they
contain the desired properties.
[0354] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue. Other insertional variants of the
antibody molecule include the fusion to the N- or C-terminus of the
antibody to an enzyme (e.g. for ADEPT) or a polypeptide which
increases the serum half-life of the antibody.
[0355] b) Glycosylation Variants
[0356] In certain embodiments, an antibody provided herein is
altered to increase or decrease the extent to which the antibody is
glycosylated. Addition or deletion of glycosylation sites to an
antibody may be conveniently accomplished by altering the amino
acid sequence such that one or more glycosylation sites is created
or removed.
[0357] Where the antibody comprises an Fc region, the carbohydrate
attached thereto may be altered. Native antibodies produced by
mammalian cells typically comprise a branched, biantennary
oligosaccharide that is generally attached by an N-linkage to
Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al.
TIBTECH 15:26-32 (1997). The oligosaccharide may include various
carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc),
galactose, and sialic acid, as well as a fucose attached to a
GlcNAc in the "stem" of the biantennary oligosaccharide structure.
In some embodiments, modifications of the oligosaccharide in an
antibody of the invention may be made in order to create antibody
variants with certain improved properties.
[0358] In one embodiment, antibody variants are provided having a
carbohydrate structure that lacks fucose attached (directly or
indirectly) to an Fc region. For example, the amount of fucose in
such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65%
or from 20% to 40%. The amount of fucose is determined by
calculating the average amount of fucose within the sugar chain at
Asn297, relative to the sum of all glycostructures attached to Asn
297 (e. g. complex, hybrid and high mannose structures) as measured
by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for
example. Asn297 refers to the asparagine residue located at about
position 297 in the Fc region (Eu numbering of Fc region residues);
however, Asn297 may also be located about +3 amino acids upstream
or downstream of position 297, i.e., between positions 294 and 300,
due to minor sequence variations in antibodies. Such fucosylation
variants may have improved ADCC function. See, e.g., US Patent
Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621
(Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to
"defucosylated" or "fucose-deficient" antibody variants include: US
2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US
2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US
2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO
2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki
et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al.
Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of
producing defucosylated antibodies include Lec13 CHO cells
deficient in protein fucosylation (Ripka et al. Arch. Biochem.
Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 A1,
Presta, L; and WO 2004/056312 A1, Adams et al., especially at
Example 11), and knockout cell lines, such as
alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,
e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda,
Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and
WO2003/085107).
[0359] Antibodies variants are further provided with bisected
oligosaccharides, e.g., in which a biantennary oligosaccharide
attached to the Fc region of the antibody is bisected by GlcNAc.
Such antibody variants may have reduced fucosylation and/or
improved ADCC function. Examples of such antibody variants are
described, e.g., in WO 2003/011878 (Jean-Mairet et al.); U.S. Pat.
No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.).
Antibody variants with at least one galactose residue in the
oligosaccharide attached to the Fc region are also provided. Such
antibody variants may have improved CDC function. Such antibody
variants are described, e.g., in WO 1997/30087 (Patel et al.); WO
1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
[0360] c) Fc Region Variants
[0361] In certain embodiments, one or more amino acid modifications
may be introduced into the Fc region of an antibody provided
herein, thereby generating an Fc region variant. The Fc region
variant may comprise a human Fc region sequence (e.g., a human
IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid
modification (e.g. a substitution) at one or more amino acid
positions.
[0362] In certain embodiments, the invention contemplates an
antibody variant that possesses some but not all effector
functions, which make it a desirable candidate for applications in
which the half life of the antibody in vivo is important yet
certain effector functions (such as complement and ADCC) are
unnecessary or deleterious. In vitro and/or in vivo cytotoxicity
assays can be conducted to confirm the reduction/depletion of CDC
and/or ADCC activities. For example, Fc receptor (FcR) binding
assays can be conducted to ensure that the antibody lacks
Fc.gamma.R binding (hence likely lacking ADCC activity), but
retains FcRn binding ability. The primary cells for mediating ADCC,
NK cells, express Fc(RIII only, whereas monocytes express Fc(RI,
Fc(RII and Fc(RIII. FcR expression on hematopoietic cells is
summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.
Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays
to assess ADCC activity of a molecule of interest is described in
U.S. Pat. No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat'l
Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc.
Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337
(see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)).
Alternatively, non-radioactive assays methods may be employed (see,
for example, ACTI.TM. non-radioactive cytotoxicity assay for flow
cytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox
96.RTM. non-radioactive cytotoxicity assay (Promega, Madison,
Wis.). Useful effector cells for such assays include peripheral
blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of
interest may be assessed in vivo, e.g., in a animal model such as
that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA
95:652-656 (1998). C1q binding assays may also be carried out to
confirm that the antibody is unable to bind C1q and hence lacks CDC
activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879
and WO 2005/100402. To assess complement activation, a CDC assay
may be performed (see, for example, Gazzano-Santoro et al., J.
Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood
101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood
103:2738-2743 (2004)). FcRn binding and in vivo clearance/half life
determinations can also be performed using methods known in the art
(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769
(2006)).
[0363] Antibodies with reduced effector function include those with
substitution of one or more of Fc region residues 238, 265, 269,
270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants
include Fc mutants with substitutions at two or more of amino acid
positions 265, 269, 270, 297 and 327, including the so-called
"DANA" Fc mutant with substitution of residues 265 and 297 to
alanine (U.S. Pat. No. 7,332,581).
[0364] Certain antibody variants with improved or diminished
binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056;
WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604
(2001).)
[0365] In certain embodiments, an antibody variant comprises an Fc
region with one or more amino acid substitutions which improve
ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the
Fc region (EU numbering of residues).
[0366] In some embodiments, alterations are made in the Fc region
that result in altered (i.e., either improved or diminished) C1q
binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as
described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et
al. J. Immunol. 164: 4178-4184 (2000).
[0367] Antibodies with increased half lives and improved binding to
the neonatal Fc receptor (FcRn), which is responsible for the
transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.
117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are
described in US2005/0014934A1 (Hinton et al.). Those antibodies
comprise an Fc region with one or more substitutions therein which
improve binding of the Fc region to FcRn. Such Fc variants include
those with substitutions at one or more of Fc region residues: 238,
256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360,
362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc
region residue 434 (U.S. Pat. No. 7,371,826).
[0368] See also Duncan & Winter, Nature 322:738-40 (1988); U.S.
Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351
concerning other examples of Fc region variants.
[0369] d) Cysteine Engineered Antibody Variants
[0370] In certain embodiments, it may be desirable to create
cysteine engineered antibodies, e.g., "thioMAbs," in which one or
more residues of an antibody are substituted with cysteine
residues. In particular embodiments, the substituted residues occur
at accessible sites of the antibody. By substituting those residues
with cysteine, reactive thiol groups are thereby positioned at
accessible sites of the antibody and may be used to conjugate the
antibody to other moieties, such as drug moieties or linker-drug
moieties, to create an immunoconjugate, as described further
herein. In certain embodiments, any one or more of the following
residues may be substituted with cysteine: V205 (Kabat numbering)
of the light chain; A118 (EU numbering) of the heavy chain; and
5400 (EU numbering) of the heavy chain Fc region. Cysteine
engineered antibodies may be generated as described, e.g., in U.S.
Pat. No. 7,521,541.
[0371] e) Antibody Derivatives
[0372] In certain embodiments, an antibody provided herein may be
further modified to contain additional nonproteinaceous moieties
that are known in the art and readily available. The moieties
suitable for derivatization of the antibody include but are not
limited to water soluble polymers. Non-limiting examples of water
soluble polymers include, but are not limited to, polyethylene
glycol (PEG), copolymers of ethylene glycol/propylene glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl
pyrrolidone, poly-1, 3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride copolymer, polyaminoacids (either
homopolymers or random copolymers), and dextran or poly(n-vinyl
pyrrolidone)polyethylene glycol, propropylene glycol homopolymers,
prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated
polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
Polyethylene glycol propionaldehyde may have advantages in
manufacturing due to its stability in water. The polymer may be of
any molecular weight, and may be branched or unbranched. The number
of polymers attached to the antibody may vary, and if more than one
polymer are attached, they can be the same or different molecules.
In general, the number and/or type of polymers used for
derivatization can be determined based on considerations including,
but not limited to, the particular properties or functions of the
antibody to be improved, whether the antibody derivative will be
used in a therapy under defined conditions, etc.
[0373] In another embodiment, conjugates of an antibody and
nonproteinaceous moiety that may be selectively heated by exposure
to radiation are provided. In one embodiment, the nonproteinaceous
moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA
102: 11600-11605 (2005)). The radiation may be of any wavelength,
and includes, but is not limited to, wavelengths that do not harm
ordinary cells, but which heat the nonproteinaceous moiety to a
temperature at which cells proximal to the
antibody-nonproteinaceous moiety are killed.
B. Recombinant Methods and Compositions
[0374] Antibodies may be produced using recombinant methods and
compositions, e.g., as described in U.S. Pat. No. 4,816,567. In one
embodiment, isolated nucleic acid encoding an anti-OX40 antibody
and/or anti-PDL1 antibody described herein is provided. Such
nucleic acid may encode an amino acid sequence comprising the VL
and/or an amino acid sequence comprising the VH of the antibody
(e.g., the light and/or heavy chains of the antibody). In a further
embodiment, one or more vectors (e.g., expression vectors)
comprising such nucleic acid are provided. In a further embodiment,
a host cell comprising such nucleic acid is provided. In one such
embodiment, a host cell comprises (e.g., has been transformed
with): (1) a vector comprising a nucleic acid that encodes an amino
acid sequence comprising the VL of the antibody and an amino acid
sequence comprising the VH of the antibody, or (2) a first vector
comprising a nucleic acid that encodes an amino acid sequence
comprising the VL of the antibody and a second vector comprising a
nucleic acid that encodes an amino acid sequence comprising the VH
of the antibody. In one embodiment, the host cell is eukaryotic,
e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0,
NS0, Sp20 cell). In one embodiment, a method of making an anti-OX40
antibody and/or anti-PDL1 antibody is provided, wherein the method
comprises culturing a host cell comprising a nucleic acid encoding
the antibody, as provided above, under conditions suitable for
expression of the antibody, and optionally recovering the antibody
from the host cell (or host cell culture medium).
[0375] For recombinant production of an anti-OX40 antibody and/or
anti-PDL1 antibody, nucleic acid encoding an antibody, e.g., as
described above, is isolated and inserted into one or more vectors
for further cloning and/or expression in a host cell. Such nucleic
acid may be readily isolated and sequenced using conventional
procedures (e.g., by using oligonucleotide probes that are capable
of binding specifically to genes encoding the heavy and light
chains of the antibody).
[0376] Suitable host cells for cloning or expression of
antibody-encoding vectors include prokaryotic or eukaryotic cells
described herein. For example, antibodies may be produced in
bacteria, in particular when glycosylation and Fc effector function
are not needed. For expression of antibody fragments and
polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237,
5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular
Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.,
2003), pp. 245-254, describing expression of antibody fragments in
E. coli.) After expression, the antibody may be isolated from the
bacterial cell paste in a soluble fraction and can be further
purified.
[0377] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for antibody-encoding vectors, including fungi and yeast strains
whose glycosylation pathways have been "humanized," resulting in
the production of an antibody with a partially or fully human
glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414
(2004), and Li et al., Nat. Biotech. 24:210-215 (2006).
[0378] Suitable host cells for the expression of glycosylated
antibody are also derived from multicellular organisms
(invertebrates and vertebrates). Examples of invertebrate cells
include plant and insect cells. Numerous baculoviral strains have
been identified which may be used in conjunction with insect cells,
particularly for transfection of Spodoptera frugiperda cells.
[0379] Plant cell cultures can also be utilized as hosts. See,
e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978,
and 6,417,429 (describing PLANTIBODIES.TM. technology for producing
antibodies in transgenic plants).
[0380] Vertebrate cells may also be used as hosts. For example,
mammalian cell lines that are adapted to grow in suspension may be
useful. Other examples of useful mammalian host cell lines are
monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic
kidney line (293 or 293 cells as described, e.g., in Graham et al.,
J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse
sertoli cells (TM4 cells as described, e.g., in Mather, Biol.
Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African
green monkey kidney cells (VERO-76); human cervical carcinoma cells
(HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL
3A); human lung cells (W138); human liver cells (Hep G2); mouse
mammary tumor (MMT 060562); TRI cells, as described, e.g., in
Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5
cells; and FS4 cells. Other useful mammalian host cell lines
include Chinese hamster ovary (CHO) cells, including DHFR CHO cells
(Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and
myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of
certain mammalian host cell lines suitable for antibody production,
see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248
(B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268
(2003).
C. Assays
[0381] Anti-OX40 antibodies and/or anti-PDL1 antibodies provided
herein may be identified, screened for, or characterized for their
physical/chemical properties and/or biological activities by
various assays known in the art.
[0382] 1. Binding Assays and Other Assays
[0383] In one aspect, an antibody of the invention is tested for
its antigen binding activity, e.g., by known methods such as ELISA,
Western blot, etc. OX40 or PDL1 binding may be determined using
methods known in the art and exemplary methods are disclosed
herein. In one embodiment, binding is measured using
radioimmunoassay. In an exemplary radioimmunassay, OX40 antibody is
iodinated, and competition reaction mixtures are prepared
containing a fixed concentration of iodinated antibody and
decreasing concentrations of serially diluted, unlabeled OZ X40
antibody. Cells expressing OX40 (e.g., BT474 cells stably
transfected with human OX40) are added to the reaction mixture.
Following an incubation, cells are washed to separate the free
iodinated OX40 antibody from the OX40 antibody bound to the cells.
Level of bound iodinated OX40 antibody is determined, e.g., by
counting radioactivity associated with cells, and binding affinity
determined using standard methods. In another embodiment, ability
of OX40 antibody to bind to surface-expressed OX40 (e.g., on T cell
subsets) is assessed using flow cytometry. Peripheral white blood
cells are obtained (e.g., from human, cynomolgus monkey, rat or
mouse) and cells are blocked with serum. Labeled OX40 antibody is
added in serial dilutions, and T cells are also stained to identify
T cell subsets (using methods known in the art). Following
incubation of the samples and washing, the cells are sorted using
flow cytometer, and data analyzed using methods well known in the
art. In another embodiment, OX40 binding may be analyzed using
surface plasmon resonance. An exemplary surface plasmon resonance
method is exemplified in the Examples.
[0384] In another aspect, competition assays may be used to
identify an antibody that competes with any of the anti-OX40
antibodies disclosed herein for binding to OX40. In certain
embodiments, such a competing antibody binds to the same epitope
(e.g., a linear or a conformational epitope) that is bound by any
of the anti-OX40 antibodies disclosed herein. Detailed exemplary
methods for mapping an epitope to which an antibody binds are
provided in Morris (1996) "Epitope Mapping Protocols," in Methods
in Molecular Biology vol. 66 (Humana Press, Totowa, N.J.). A
competition assay is exemplified in the Examples.
[0385] In an exemplary competition assay, immobilized OX40 is
incubated in a solution comprising a first labeled antibody that
binds to OX40 (e.g., mab 1A7.gr.1, mab 3C8.gr5) and a second
unlabeled antibody that is being tested for its ability to compete
with the first antibody for binding to OX40. The second antibody
may be present in a hybridoma supernatant. As a control,
immobilized OX40 is incubated in a solution comprising the first
labeled antibody but not the second unlabeled antibody. After
incubation under conditions permissive for binding of the first
antibody to OX40, excess unbound antibody is removed, and the
amount of label associated with immobilized OX40 is measured. If
the amount of label associated with immobilized OX40 is
substantially reduced in the test sample relative to the control
sample, then that indicates that the second antibody is competing
with the first antibody for binding to OX40. See Harlow and Lane
(1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.). It will be appreciated that
similar techniques may be used to identify anti-PDL1
antibodies.
[0386] 2. Activity Assays
[0387] In one aspect, assays are provided for identifying anti-OX40
antibodies thereof having biological activity. Biological activity
may include, e.g., binding OX40 (e.g., binding human and/or
cynomolgus OX40), increasing OX40-mediated signal transduction
(e.g., increasing NFkB-mediated transcription), depleting cells
that express human OX40 (e.g., T cells), depleting cells that
express human OX40 by ADCC and/or phagocytosis, enhancing T
effector cell function (e.g., CD4+ effector T cell), e.g., by
increasing effector T cell proliferation and/or increasing cytokine
production (e.g., gamma interferon) by effector T cells, enhancing
memory T cell function (e.g., CD4+ memory T cell), e.g., by
increasing memory T cell proliferation and/or increasing cytokine
production by memory T cells (e.g., gamma interferon), inhibiting
regulatory T cell function (e.g., by decreasing Treg suppression of
effector T cell function (e.g., CD4+ effector T cell function),
binding human effector cells. Antibodies having such biological
activity in vivo and/or in vitro are also provided.
[0388] In certain embodiments, an antibody of the invention is
tested for such biological activity.
[0389] T cell costimulation may be assayed using methods known in
the art and exemplary methods are disclosed herein. For example, T
cells (e.g., memory or effector T cells) may be obtained from
peripheral white blood cells (e.g., isolated from human whole blood
using Ficoll gradient centrifugation). Memory T cells (e.g., CD4+
memory T cells) or effector T cells (e.g. CD4+ Teff cells) may be
isolated from PBMC using methods known in the art. For example, the
Miltenyi CD4+ memory T cell isolation kit or Miltenyi naive CD4+ T
cell isolation kit may be used. Isolated T cells are cultured in
the presence of antigen presenting cells (e.g., irradiated L cells
that express CD32 and CD80), and activated by addition of anti-CD3
antibody in the presence or absence of OX40 agonist antibody.
Effect of agonist OX40 antibody of T cell proliferation may be
measured using methods well known in the art. For example, the
CellTiter Glo kit (Promega) may be used, and results read on a
Multilabel Reader (Perkin Elmer). Effect of agonist OX40 antibody
on T cell function may also be determined by analysis of cytokines
produced by the T cell. In one embodiment, production of interferon
gamma by CD4+ T cells is determined, e.g., by measurement of
interferon gamma in cell culture supernatant. Methods for measuring
interferon gamma are well-known in the art.
[0390] Treg cell function may be assayed using methods known in the
art and exemplary methods are disclosed herein. In one example, the
ability of Treg to suppress effector T cell proliferation is
assayed. T cells are isolated from human whole blood using methods
known in the art (e.g., isolating memory T cells or naive T cells).
Purified CD4+ naive T cells are labeled (e.g., with CFSE) and
purified Treg cells are labeled with a different reagent.
Irradiated antigen presenting cells (e.g., L cells expressing CD32
and CD80) are co-cultured with the labeled purified naive CD4+ T
cells and purified Tregs. The co-cultures are activated using
anti-CD3 antibody and tested in the presence or absence of agonist
OX40 antibody. Following a suitable time (e.g., 6 days of
coculture), level of CD4+ naive T cell proliferation is tracked by
dye dilution in reduced label staining (e.g., reduced CFSE label
staining) using FACS analysis.
[0391] OX40 signaling may be assayed using methods well known in
the art and exemplary methods are disclosed herein. In one
embodiment, transgenic cells are generated that express human OX40
and a reporter gene comprising the NFkB promoter fused to a
reporter gene (e.g., beta luciferase). Addition of OX40 agonist
antibody to the cells results in increased NFkB transcription,
which is detected using an assay for the reporter gene.
[0392] Phagocytosis may be assayed, e.g., by using monocyte-derived
macrophages, or U937 cells (a human histiocytic lymphoma cells line
with the morphology and characteristics of mature macrophages).
OX40 expressing cells are added to the monocyte-derived macrophages
or U937 cells in the presence or absence of anti-OX40 agonist
antibody. Following culturing of the cells for a suitable period of
time, the percentage of phagocytosis is determined by examining
percentage of cells that double stain for markers of 1) the
macrophage or U937 cell and 2) the OX40 expressing cell, and
dividing this by the total number of cells that show markers of the
OX40 expressing cell (e.g., GFP). Analysis may be done by flow
cytometry. In another embodiment, analysis may be done by
fluorescent microscopy analysis.
[0393] ADCC may be assayed, e.g., using methods well known in the
art. Exemplary methods are described in the definition section and
an exemplary assay is disclosed in the Examples. In some
embodiments, level of OX40 is characterized on an OX40 expressing
cell that is used for testing in an ADCC assay. The cell may be
stained with a detectably labeled anti-OX40 antibody (e.g., PE
labeled), then level of fluorescence determined using flow
cytometry, and results presented as median fluorescence intensity
(MFI). In another embodiment, ADCC may be analyzed by CellTiter Glo
assay kit and cell viability/cytotoxicity may be determined by
chemioluminescence.
[0394] The binding affinities of various antibodies to
Fc.gamma.RIA, Fc.gamma.RIIA, Fc.gamma.RIIB, and two allotypes of
Fc.gamma.RIIIA (F158 and V158) may be measured in ELISA-based
ligand-binding assays using the respective recombinant Fc.gamma.
receptors. Purified human Fc.gamma. receptors are expressed as
fusion proteins containing the extracellular domain of the receptor
.gamma. chain linked to a Gly/6.times.His/glutathione S-transferase
(GST) polypeptide tag at the C-terminus. The binding affinities of
antibodies to those human Fc.gamma. receptors are assayed as
follows. For the low-affinity receptors, i.e. Fc.gamma.RIIA
(CD32A), Fc.gamma.RIIB (CD32B), and the two allotypes of
Fc.gamma.RIIIA (CD16), F-158 and V-158, antibodies may be tested as
multimers by cross-linking with a F(ab')2 fragment of goat
anti-human kappa chain (ICN Biomedical; Irvine, Calif.) at an
approximate molar ratio of 1:3 antibody:cross-linking F(ab').sub.2.
Plates are coated with an anti-GST antibody (Genentech) and blocked
with bovine serum albumin (BSA). After washing with
phosphate-buffered saline (PBS) containing 0.05% Tween-20 with an
ELx405.TM. plate washer (Biotek Instruments; Winooski, Vt.),
Fc.gamma. receptors are added to the plate at 25 ng/well and
incubated at room temperature for 1 hour. After the plates are
washed, serial dilutions of test antibodies are added as multimeric
complexes and the plates were incubated at room temperature for 2
hours. Following plate washing to remove unbound antibodies, the
antibodies bound to the Fc.gamma. receptor are detected with
horseradish peroxidase (HRP)-conjugated F(ab').sub.2 fragment of
goat anti-human F(ab').sub.2 (Jackson ImmunoResearch Laboratories;
West Grove, Pa.) followed by the addition of substrate,
tetramethylbenzidine (TMB) (Kirkegaard & Perry Laboratories;
Gaithersburg, Md.). The plates are incubated at room temperature
for 5-20 minutes, depending on the Fc.gamma. receptors tested, to
allow color development. The reaction is terminated with 1 M
H.sub.3PO.sub.4 and absorbance at 450 nm was measured with a
microplate reader (SpectraMax.RTM.190, Molecular Devices;
Sunnyvale, Calif.). Dose-response binding curves are generated by
plotting the mean absorbance values from the duplicates of antibody
dilutions against the concentrations of the antibody. Values for
the effective concentration of the antibody at which 50% of the
maximum response from binding to the Fc.gamma. receptor is detected
(EC.sub.50) were determined after fitting the binding curve with a
four-parameter equation using SoftMax Pro (Molecular Devices).
[0395] To select for antibodies which induce cell death, loss of
membrane integrity as indicated by, e.g., propidium iodide (PI),
trypan blue or 7AAD uptake may be assessed relative to control. A
PI uptake assay can be performed in the absence of complement and
immune effector cells. OX40 expressing cells are incubated with
medium alone or medium containing of the appropriate monoclonal
antibody at e.g., about 10 .mu.g/ml. The cells are incubated for a
time period (e.g., 1 or 3 days). Following each treatment, cells
are washed and aliquoted. In some embodiments, cells are aliquoted
into 35 mm strainer-capped 12.times.75 tubes (1 ml per tube, 3
tubes per treatment group) for removal of cell clumps. Tubes then
receive PI (10 .mu.g/ml). Samples may be analyzed using a
FACSCAN.TM. flow cytometer and FACSCONVERT.TM. CellQuest software
(Becton Dickinson).
[0396] Cells for use in any of the above in vitro assays include
cells or cell lines that naturally express OX40 or that have been
engineered to express OX40. Such cells include activated T cells,
Treg cells and activated memory T cells that naturally express
OX40. Such cells also include cell lines that express OX40 and cell
lines that do not normally express OX40 but have been transfected
with nucleic acid encoding OX40. Exemplary cell lines provided
herein for use in any of the above in vitro assays include
transgenic BT474 cells (a human breast cancer cell line) that
express human OX40.
[0397] Anti-PDL1 antibodies may be identified using methods known
in the art (such as ELISA, Western Blot, biological activity
assays, etc.). For example, for an anti-PDL1 antibody, the antigen
binding properties of the antibody can be evaluated in an assay
that detects the ability to bind to PDL1. In some embodiments, the
binding of the antibody may be determined by saturation binding;
ELISA; and/or competition assays (e.g. RIA's), for example. Also,
the antibody may be subjected to other biological activity assays,
e.g., in order to evaluate its effectiveness as a therapeutic. Such
assays are known in the art and depend on the target antigen and
intended use for the antibody. For example, the biological effects
of PD-L1 blockade by the antibody can be assessed in CD8+ T cells,
a lymphocytic choriomeningitis virus (LCMV) mouse model and/or a
syngeneic tumor model e.g., as described in U.S. Pat. No.
8,217,149.
[0398] To screen for antibodies which bind to a particular epitope
on the antigen of interest (e.g., those which block binding of the
anti-PDL1 antibody of the example to PD-L1), a routine
cross-blocking assay such as that described in Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and
David Lane (1988), can be performed. Alternatively, epitope
mapping, e.g. as described in Champe et al., J. Biol. Chem.
270:1388-1394 (1995), can be performed to determine whether the
antibody binds an epitope of interest.
[0399] It is understood that any of the above assays may be carried
out using an immunoconjugate of the invention in place of or in
addition to an anti-OX40 antibody and/or anti-PDL1 antibody.
[0400] It is understood that any of the above assays may be carried
out using anti-OX40 antibody and/or anti-PDL1 antibody and an
additional therapeutic agent.
D. Immunoconjugates
[0401] The invention also provides immunoconjugates comprising an
anti-OX40 antibody herein conjugated to one or more cytotoxic
agents, such as chemotherapeutic agents or drugs, growth inhibitory
agents, toxins (e.g., protein toxins, enzymatically active toxins
of bacterial, fungal, plant, or animal origin, or fragments
thereof), or radioactive isotopes.
[0402] In one embodiment, an immunoconjugate is an antibody-drug
conjugate (ADC) in which an antibody is conjugated to one or more
drugs, including but not limited to a maytansinoid (see U.S. Pat.
Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); an
auristatin such as monomethylauristatin drug moieties DE and DF
(MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588, and
7,498,298); a dolastatin; a calicheamicin or derivative thereof
(see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285,
5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al.,
Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res.
58:2925-2928 (1998)); an anthracycline such as daunomycin or
doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523
(2006); Jeffrey et al., Bioorganic & Med. Chem. Letters
16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005);
Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000);
Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532
(2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S.
Pat. No. 6,630,579); methotrexate; vindesine; a taxane such as
docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a
trichothecene; and CC1065.
[0403] In another embodiment, an immunoconjugate comprises an
antibody as described herein conjugated to an enzymatically active
toxin or fragment thereof, including but not limited to diphtheria
A chain, nonbinding active fragments of diphtheria toxin, exotoxin
A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A
chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins,
dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and
PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes.
[0404] In another embodiment, an immunoconjugate comprises an
antibody as described herein conjugated to a radioactive atom to
form a radioconjugate. A variety of radioactive isotopes are
available for the production of radioconjugates. Examples include
At.sup.211, I.sup.131, I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188,
Sm.sup.153, Bi.sup.212, P.sup.32, Pb.sup.212 and radioactive
isotopes of Lu. When the radioconjugate is used for detection, it
may comprise a radioactive atom for scintigraphic studies, for
example tc99m or I123, or a spin label for nuclear magnetic
resonance (NMR) imaging (also known as magnetic resonance imaging,
mri), such as iodine-123 again, iodine-131, indium-111,
fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium,
manganese or iron.
[0405] Conjugates of an antibody and cytotoxic agent may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate
(SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters
(such as dimethyl adipimidate HCl), active esters (such as
disuccinimidyl suberate), aldehydes (such as glutaraldehyde),
bis-azido compounds (such as his (p-azidobenzoyl) hexanediamine),
bis-diazonium derivatives (such as
bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as
toluene 2,6-diisocyanate), and bis-active fluorine compounds (such
as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin
immunotoxin can be prepared as described in Vitetta et al., Science
238:1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026. The linker may be
a "cleavable linker" facilitating release of a cytotoxic drug in
the cell. For example, an acid-labile linker, peptidase-sensitive
linker, photolabile linker, dimethyl linker or disulfide-containing
linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No.
5,208,020) may be used.
[0406] The immunuoconjugates or ADCs herein expressly contemplate,
but are not limited to such conjugates prepared with cross-linker
reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS,
LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS,
sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and
sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which
are commercially available (e.g., from Pierce Biotechnology, Inc.,
Rockford, Ill., U.S.A).
E. Methods and Compositions for Diagnostics and Detection
[0407] In certain embodiments, any of the anti-OX40 antibodies
provided herein is useful for detecting the presence of OX40 in a
biological sample. The term "detecting" as used herein encompasses
quantitative or qualitative detection. In certain embodiments, a
biological sample comprises a cell or tissue, such as a sample of a
tumor (e.g., NSCLC or breast tumor).
[0408] In one embodiment, an anti-OX40 antibody for use in a method
of diagnosis or detection is provided. In a further aspect, a
method of detecting the presence of OX40 in a biological sample is
provided. In certain embodiments, the method comprises contacting
the biological sample with an anti-OX40 antibody as described
herein under conditions permissive for binding of the anti-OX40
antibody to OX40, and detecting whether a complex is formed between
the anti-OX40 antibody and OX40. Such method may be an in vitro or
in vivo method. In one embodiment, an anti-OX40 antibody is used to
select subjects eligible for therapy with an anti-OX40 antibody,
e.g. where OX40 is a biomarker for selection of patients.
[0409] In some embodiments, the anti-OX40 antibody for use in a
method of diagnosis or detection is an anti-human OX40 antibody
comprising at least one, two, three, four, five, or six HVRs
selected from (a) HVR-H1 comprising the amino acid sequence of SEQ
ID NO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:3; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4;
(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f)
HVR-L3 comprising the amino acid sequence of SEQ ID NO:7. In some
embodiments, the anti-OX40 antibody comprises (a) a VH domain
comprising at least one, at least two, or all three VH HVR
sequences selected from (i) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:2, (ii) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:3, and (iii) HVR-H3 comprising an amino acid
sequence selected from SEQ ID NO:4; and (b) a VL domain comprising
at least one, at least two, or all three VL HVR sequences selected
from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5,
(ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6, and
(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:7. In
some embodiments, the OX40 antibody comprises (a) HVR-H1 comprising
the amino acid sequence of SEQ ID NO:2; (b) HVR-H2 comprising the
amino acid sequence of SEQ ID NO:3; (c) HVR-H3 comprising the amino
acid sequence of SEQ ID NO:4; (d) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:6; and (f) HVR-L3 comprising an amino acid
sequence selected from SEQ ID NO:7. In some embodiments, the
antibody comprises a heavy chain variable domain (VH) sequence
having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100% sequence identity to the amino acid sequence of SEQ ID
NO:180. In certain embodiments, a VH sequence having at least 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the reference sequence, but an anti-human
OX40 agonist antibody comprising that sequence retains the ability
to bind to OX40. In certain embodiments, a total of 1 to 10 amino
acids have been substituted, inserted and/or deleted in SEQ ID
NO:180. In certain embodiments, substitutions, insertions, or
deletions occur in regions outside the HVRs (i.e., in the FRs).
Optionally, the anti-human OX40 agonist antibody comprises the VH
sequence in SEQ ID NO:180, including post-translational
modifications of that sequence. In a particular embodiment, the VH
comprises one, two or three HVRs selected from: (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO:2, (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO:3, and (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:4. In some
embodiments, the antibody comprises a light chain variable domain
(VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or 100% sequence identity to the amino acid sequence of SEQ ID
NO:179. In certain embodiments, a VL sequence having at least 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the reference sequence, but an anti-human
OX40 agonist antibody comprising that sequence retains the ability
to bind to OX40. In certain embodiments, a total of 1 to 10 amino
acids have been substituted, inserted and/or deleted in SEQ ID NO:
179. In certain embodiments, the substitutions, insertions, or
deletions occur in regions outside the HVRs (i.e., in the FRs).
Optionally, the anti-human OX40 agonist antibody comprises the VL
sequence in SEQ ID NO: 179, including post-translational
modifications of that sequence. In a particular embodiment, the VL
comprises one, two or three HVRs selected from (a) HVR-L1
comprising the amino acid sequence of SEQ ID NO:5; (b) HVR-L2
comprising the amino acid sequence of SEQ ID NO:6; and (c) HVR-L3
comprising the amino acid sequence of SEQ ID NO:7.
[0410] In some embodiments, the anti-OX40 antibody used in the
method of diagnosis or detection is an anti-human OX40 antibody
comprising at least one, two, three, four, five, or six HVRs
selected from (a) HVR-H1 comprising the amino acid sequence of SEQ
ID NO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:30; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:31; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:37; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:39; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:42. In some embodiments, the anti-OX40 antibody comprises (a) a
VH domain comprising at least one, at least two, or all three VH
HVR sequences selected from (i) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:29, (ii) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:30, and (iii) HVR-H3 comprising an amino acid
sequence selected from SEQ ID NO:31; and (b) a VL domain comprising
at least one, at least two, or all three VL HVR sequences selected
from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37,
(ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:39, and
(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:42. In
some embodiments, the anti-OX40 antibody comprises (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO:29; (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO:30; (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:31; (d) HVR-L1
comprising the amino acid sequence of SEQ ID NO:37; (e) HVR-L2
comprising the amino acid sequence of SEQ ID NO:39; and (f) HVR-L3
comprising an amino acid sequence selected from SEQ ID NO:42. In
some embodiment, the anti-OX40 antibody comprises a heavy chain
variable domain (VH) sequence having at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the
amino acid sequence of SEQ ID NO:182. In certain embodiments, a VH
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity contains substitutions (e.g., conservative
substitutions), insertions, or deletions relative to the reference
sequence, but an anti-human OX40 agonist antibody comprising that
sequence retains the ability to bind to OX40. In certain
embodiments, a total of 1 to 10 amino acids have been substituted,
inserted and/or deleted in SEQ ID NO:182. In certain embodiments,
substitutions, insertions, or deletions occur in regions outside
the HVRs (i.e., in the FRs). Optionally, the anti-human OX40
agonist antibody comprises the VH sequence in SEQ ID NO:182,
including post-translational modifications of that sequence. In a
particular embodiment, the VH comprises one, two or three HVRs
selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ
ID NO:29, (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:30, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:31. In some embodiments, the anti-OX40 antibody comprises a
light chain variable domain (VL) having at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the
amino acid sequence of SEQ ID NO:181. In certain embodiments, a VL
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity contains substitutions (e.g., conservative
substitutions), insertions, or deletions relative to the reference
sequence, but an anti-human OX40 agonist antibody comprising that
sequence retains the ability to bind to OX40. In certain
embodiments, a total of 1 to 10 amino acids have been substituted,
inserted and/or deleted in SEQ ID NO: 181. In certain embodiments,
the substitutions, insertions, or deletions occur in regions
outside the HVRs (i.e., in the FRs). Optionally, the anti-human
OX40 agonist antibody comprises the VL sequence in SEQ ID NO: 181,
including post-translational modifications of that sequence. In a
particular embodiment, the VL comprises one, two or three HVRs
selected from (a) HVR-L1 comprising the amino acid sequence of SEQ
ID NO:37; (b) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:39; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:42.
[0411] In some embodiments, the anti-OX40 antibody comprises a VH
sequence of SEQ ID NO: 180. In some embodiments, the anti-OX40
antibody comprises a VL sequence of SEQ ID NO: 179. In some
embodiments, the anti-OX40 antibody comprises a VH sequence of SEQ
ID NO:180 and a VL sequence of SEQ ID NO: 179. In some embodiments,
the anti-OX40 antibody comprises a VH sequence of SEQ ID NO: 182.
In some embodiments, the anti-OX40 antibody comprises a VL sequence
of SEQ ID NO: 181. In some embodiments, the anti-OX40 antibody
comprises a VH sequence of SEQ ID NO:182 and a VL sequence of SEQ
ID NO: 181.
[0412] Exemplary disorders that may be diagnosed using an antibody
of the invention include cancer.
[0413] In certain embodiments, labeled anti-OX40 antibodies are
provided. Labels include, but are not limited to, labels or
moieties that are detected directly (such as fluorescent,
chromophoric, electron-dense, chemiluminescent, and radioactive
labels), as well as moieties, such as enzymes or ligands, that are
detected indirectly, e.g., through an enzymatic reaction or
molecular interaction. Exemplary labels include, but are not
limited to, the radioisotopes .sup.32P, .sup.14C, .sup.125I,
.sup.3H, and .sup.131I, fluorophores such as rare earth chelates or
fluorescein and its derivatives, rhodamine and its derivatives,
dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and
bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin,
2,3-dihydrophthalazinediones, horseradish peroxidase (HRP),
alkaline phosphatase, .beta.-galactosidase, glucoamylase, lysozyme,
saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and
glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as
uricase and xanthine oxidase, coupled with an enzyme that employs
hydrogen peroxide to oxidize a dye precursor such as HRP,
lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,
bacteriophage labels, stable free radicals, and the like.
[0414] In one aspect, the invention provides diagnostic methods,
e.g. for identifying a cancer patient who is likely to respond to
treatment with an anti-human OX40 agonist antibody.
[0415] In some embodiments, methods are provided for identifying
patients who are likely to respond to treatment with anti-human
OX40 agonist antibody, the methods comprising (i) determining
presence or absence or amount (e.g., number per given sample size)
of cells expressing FcR in a sample of cancer from the patient, and
(ii) identifying the patient as likely to respond if the sample
comprises cells expressing FcR (e.g., high number of cells
expressing FcR). Methods for detecting cells that express FcR are
well known in the art, including, e.g., by IHC. In some
embodiments, FcR is Fc.gamma.R. In some embodiments, FcR is an
activating Fc.gamma.R. In some embodiments, the cancer is any
cancer described herein. In some embodiments, the cancer is
non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma,
melanoma, breast carcinoma (e.g. triple-negative breast cancer),
gastric cancer, colorectal cancer (CRC), or hepatocellular
carcinoma. In some embodiments, the method is an in vitro method.
In some embodiments, the methods further comprise (iii)
recommending treatment with the anti-human OX40 agonist antibody
(e.g., any of the anti-human OX40 agonist antibodies described
herein). In some embodiments, the methods further comprise (iv)
treating the patient with the anti-human OX40 agonist antibody.
[0416] In some embodiments, methods are provided for identifying
patients who are likely to respond to treatment with anti-human
OX40 agonist antibody, the methods comprising (i) determining
presence or absence or amount (e.g., number per given sample size)
of human effector cells (e.g., infiltrating effector cells) in a
sample of cancer from the patient, and (ii) identifying the patient
as likely to respond if the sample comprises effector cells (e.g.,
high number of effector cells). Methods for detecting infiltrating
human effector cells are well known in the art, including, e.g., by
IHC. In some embodiments, human effector cells are one or more of
NK cells, macrophages, monocytes. In some embodiments, the effector
cells express activating Fc.gamma.R. In some embodiments, the
method is an in vitro method. In some embodiments, the cancer is
any cancer described herein. In some embodiments, the cancer is
non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma,
melanoma, breast carcinoma (e.g. triple-negative breast cancer),
gastric cancer, colorectal cancer (CRC), or hepatocellular
carcinoma. In some embodiments, the methods further comprise (iii)
recommending treatment with the anti-human OX40 agonist antibody
(e.g., any of the anti-human OX40 agonist antibodies described
herein). In some embodiments, the methods further comprise (iv)
treating the patient with the anti-human OX40 agonist antibody.
[0417] Provided are methods of providing a cancer diagnosis
comprising: (i) measuring FcR expressing cells (e.g., the level or
presence or absence of or prevalence (e.g., percentage of cells
expressing FcR, e.g., by IHC) of FcR) in a sample from the patient;
(ii) diagnosing the patient as having cancer comprising FcR
biomarker (e.g., high FcR biomarker) when the sample has FcR
biomarker expression. In some embodiments, the method further
comprises (iii) selecting a therapy comprising (a) anti-human OX40
agonist antibody or (b) recommending a therapy comprising
anti-human OX40 agonist antibody for the patient. In some
embodiments, the method is an in vitro method.
[0418] Provided are methods of providing a cancer diagnosis
comprising: (i) measuring human effector cells (e.g., the level or
presence or absence of or prevalence (e.g., percentage of human
effector cells) of human effector cells) in a sample from the
patient; (ii) diagnosing the patient as having cancer comprising
human effector cells (e.g., high human effector cells) when the
sample has human effector cell biomarker. In some embodiments, the
method further comprises (iii) selecting a therapy comprising (a)
anti-human OX40 agonist antibody or (b) recommending a therapy
comprising anti-human OX40 agonist antibody for the patient. In
some embodiments, the method is an in vitro method.
[0419] Provided are methods of recommending a treatment to a cancer
patient comprising: (i) measuring FcR expressing cells (e.g., the
level or presence or absence of or prevalence (e.g., percentage of
cells expressing FcR) of FcR) in a sample from the patient; (ii)
recommending treatment with an anti-human OX40 agonist antibody
when the sample has FcR expressing cells (in some embodiments, high
FcR expressing cells). In some embodiments, the method further
comprises (iii) selecting a therapy comprising anti-human OX40
agonist antibody for the patient. In some embodiments, the method
is an in vitro method.
[0420] Provided are methods of recommending a treatment to a cancer
patient comprising: (i) measuring human effector cells (e.g., the
level or presence or absence of or prevalence (e.g., percentage of
human effector cells) of human effector cells) in a sample from the
patient; (ii) recommending treatment with an anti-human OX40
agonist antibody when the sample has human effector cells (in some
embodiments, high human effector cells). In some embodiments, the
method further comprises (iii) selecting a therapy comprising
anti-human OX40 agonist antibody for the patient. In some
embodiments, the method is an in vitro method.
[0421] In some embodiments of any of the inventions provided
herein, the sample is obtained prior to treatment with anti-human
OX40 agonist antibody. In some embodiments, the sample is obtained
prior to treatment with a cancer medicament. In some embodiments,
the sample is obtained after the cancer has metastasized. In some
embodiments, the sample is formalin fixed and paraffin embedded
(FFPE). In some embodiments, the sample is of a biopsy (e.g., a
core biopsy), a surgical specimen (e.g., a specimen from a surgical
resection), or a fine needle aspirate.
F. Pharmaceutical Formulations
[0422] Pharmaceutical formulations of an anti-OX40 antibody as
described herein are prepared by mixing such antibody having the
desired degree of purity with one or more optional pharmaceutically
acceptable carriers (Remington's Pharmaceutical Sciences 16th
edition, Osol, A. Ed. (1980)), in the form of lyophilized
formulations or aqueous solutions. Pharmaceutically acceptable
carriers are generally nontoxic to recipients at the dosages and
concentrations employed, and include, but are not limited to:
buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyldimethylbenzyl ammonium chloride; hexamethonium
chloride; benzalkonium chloride; benzethonium chloride; phenol,
butyl or benzyl alcohol; alkyl parabens such as methyl or propyl
paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and
m-cresol); low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, histidine,
arginine, or lysine; monosaccharides, disaccharides, and other
carbohydrates including glucose, mannose, or dextrins; chelating
agents such as EDTA; sugars such as sucrose, mannitol, trehalose or
sorbitol; salt-forming counter-ions such as sodium; metal complexes
(e.g. Zn-protein complexes); and/or non-ionic surfactants such as
polyethylene glycol (PEG). Exemplary pharmaceutically acceptable
carriers herein further include insterstitial drug dispersion
agents such as soluble neutral-active hyaluronidase glycoproteins
(sHASEGP), for example, human soluble PH-20 hyaluronidase
glycoproteins, such as rHuPH20 (HYLENEX.RTM., Baxter International,
Inc.). Certain exemplary sHASEGPs and methods of use, including
rHuPH20, are described in US Patent Publication Nos. 2005/0260186
and 2006/0104968. In one aspect, a sHASEGP is combined with one or
more additional glycosaminoglycanases such as chondroitinases.
[0423] In some embodiments, a "histidine buffer" is a buffer
comprising histidine ions. Examples of histidine buffers include
histidine chloride, histidine acetate, histidine phosphate,
histidine sulfate. The preferred histidine buffer identified in the
examples herein was found to be histidine acetate. In the preferred
embodiment, the histidine acetate buffer is prepared by titrating
L-histidine (free base, solid) with acetic acid (liquid). In some
embodiments, the histidine buffer or histidine-acetate buffer is at
pH 5.0 to 6.0, in some embodiments, pH 5.3 to 5.8.
[0424] In some embodiments, a "saccharide" herein comprises the
general composition (CH2O)n and derivatives thereof, including
monosaccharides, disaccharides, trisaccharides, polysaccharides,
sugar alcohols, reducing sugars, nonreducing sugars, etc. Examples
of saccharides herein include glucose, sucrose, trehalose, lactose,
fructose, maltose, dextran, glycerin, dextran, erythritol,
glycerol, arabitol, sylitol, sorbitol, mannitol, mellibiose,
melezitose, raffinose, mannotriose, stachyose, maltose, lactulose,
maltulose, glucitol, maltitol, lactitol, iso-maltulose, etc. In
some embodiments, the saccharide is a nonreducing disaccharide,
such as trehalose or sucrose.
[0425] In some embodiments herein, a "surfactant" refers to a
surface-active agent, preferably a nonionic surfactant. Examples of
surfactants herein include polysorbate (for example, polysorbate 20
and polysorbate 80); poloxamer (e.g. poloxamer 188); Triton; sodium
dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl
glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine;
lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-,
myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-,
linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or
isostearamidopropyl-betaine (e.g. lauroamidopropyl);
myristamidopropyl-, palmidopropyl-, or
isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or
disodium methyl oleyl-taurate; and the MONAQUAT.TM. series (Mona
Industries, Inc., Paterson, N.J.); polyethyl glycol, polypropyl
glycol, and copolymers of ethylene and propylene glycol (e.g.
Pluronics, PF68 etc); etc. In some embodiments, the surfactant is
polysorbate 20. In some embodiments, the surfactant is polysorbate
80.
[0426] Exemplary lyophilized antibody formulations are described in
U.S. Pat. No. 6,267,958. Aqueous antibody formulations include
those described in U.S. Pat. No. 6,171,586 and WO2006/044908, the
latter formulations including a histidine-acetate buffer.
[0427] The formulation herein may also contain more than one active
ingredients as necessary for the particular indication being
treated, preferably those with complementary activities that do not
adversely affect each other. For example, it may be desirable to
further provide an additional medicament (examples of which are
provided herein). Such active ingredients are suitably present in
combination in amounts that are effective for the purpose
intended.
[0428] Active ingredients may be entrapped in microcapsules
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsules and poly-(methylmethacylate) microcapsules,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980).
[0429] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules.
[0430] The formulations to be used for in vivo administration are
generally sterile. Sterility may be readily accomplished, e.g., by
filtration through sterile filtration membranes.
[0431] In some embodiments, provided herein are pharmaceutical
formulations comprising: (a) any of the anti-human OX40 agonist
antibodies described herein; (b) a histidine buffer at pH
5.0-6.0.
[0432] In some embodiments, provided herein are pharmaceutical
formulations comprising: (a) any of the anti-human OX40 agonist
antibodies described herein; (b) a histidine buffer at pH 5.0-6.0;
(c) a saccharide; and (d) a surfactant.
[0433] In some embodiments of any of the formulations, the
anti-human OX40 agonist antibody is present at a concentration
between about 10 mg/mL and about 100 mg/mL (e.g. about 15 mg/mL, 18
mg/mL, 20 mg/mL, 60 mg/mL, and 75 mg/mL). In some embodiments, the
anti-human OX40 agonist antibody is present at a concentration of
about 20 mg/mL. In some embodiments, the anti-human OX40 agonist
antibody is present at a concentration of about 50 mg/mL. In some
embodiments, the anti-human OX40 agonist antibody is present at a
concentration of about 60 mg/mL. In some embodiments, the
anti-human OX40 agonist antibody is present at a concentration of
about 70 mg/mL.
[0434] In some embodiments of any of the formulations, the
saccharide is present at a concentration of about 75 mM to about
360 mM (e.g., about 100 mM, about 120 mM, about 240 mM, about 320
mM to about 360 mM). In some embodiments, the saccharide is present
at a concentration of about 120 mM. In some embodiments, the
saccharide is present at a concentration of about 240 mM. In some
embodiments, the saccharide is present at a concentration of about
320 mM. In some embodiments, the saccharide is a disaccharide. In
some embodiments, the disaccharide is trehalose. In some
embodiments, the disaccharide is sucrose.
[0435] In some embodiments of any of the formulations, the
histidine buffer is at a concentration of about 1 mM to about 50 mM
(e.g. about 1 mM to about 25 mM). In some embodiments, the
histidine buffer is at a concentration of about 10 mM. In some
embodiments, the histidine buffer is at a concentration of about 20
mM. In some embodiments, the histidine buffer is at a concentration
of about 30 mM. In some embodiments, the histidine buffer is
histidine acetate.
[0436] In some embodiments of any of the formulations, the
surfactant is polysorbate (e.g., polysorbate 20 or polysorbate 40),
poloxamer (e.g. poloxamer 188); Triton; sodium dodecyl sulfate
(SDS); sodium laurel sulfate; or sodium octyl glycoside.
[0437] In some embodiments of any of the formulations, the
surfactant is polysorbate. In some embodiments, the polysorbate is
present at a concentration of about 0.005% to about 0.1%. In some
embodiments, the polysorbate is present at a concentration of about
0.005%. In some embodiments, the polysorbate is present at a
concentration of about 0.02%. In some embodiments, the polysorbate
is present at a concentration of about 0.04%. In some embodiments,
the polysorbate is present at a concentration of about 0.06%. In
some embodiments, the polysorbate is polysorbate 20. In some
embodiments, the polysorbate is polysorbate 80.
[0438] In some embodiments of any of the formulations, the
formulation is diluted with a diluent (e.g., 0.9% NaCl). In some
embodiments, the anti-human OX40 agonist antibody is present at a
concentration of about 1 mg/mL.
[0439] In particular, provided herein are pharmaceutical
formulations comprising (a) any of the anti-human OX40 agonist
antibodies described herein, (b) a polysorbate, wherein the
polysorbate concentration is about 0.005% to about 0.1%; and (c) a
histidine buffer (e.g., a histidine buffer at a pH between 5.0 and
6.0).
[0440] In some embodiments, the pharmaceutical formulation
comprises (a) any of the anti-human OX40 agonist antibodies
described herein (e.g., at a concentration between about 10 mg/mL
and about 100 mg/mL), (b) a polysorbate, wherein the polysorbate
concentration is about 0.02% to about 0.06%; (c) a histidine buffer
(e.g., a histidine buffer at pH 5.0 to 6.0); and a saccharide,
wherein the saccharide concentration is about 120 mM to about 320
mM. In some embodiments, the saccharide is sucrose.
[0441] In some embodiments, the pharmaceutical formulation
comprises (a) any of the anti-human OX40 agonist antibodies
described herein at a concentration between about 10 mg/mL and
about 100 mg/mL, (b) a polysorbate, wherein the polysorbate
concentration is about 0.02% to about 0.06%, wherein the
polysorbate is polysorbate 20 or polysorbate 40; (c) a histidine
acetate buffer at pH 5.0 to 6.0; and a saccharide (e.g., sucrose)
at a concentration of about 120 mM to about 320 mM.
[0442] In some embodiments, the pharmaceutical formulation
comprises (a) any of the anti-human OX40 agonist antibodies
described herein, (b) polysorbate 20, wherein the polysorbate
concentration is about 0.02% to about 0.06%; (c) a histidine
acetate buffer (e.g., a histidine acetate buffer at pH 5.0 to 6.0);
and (d) sucrose, wherein the sucrose concentration is about 120 mM
to about 320 mM.
[0443] In some embodiments, the pharmaceutical formulation
comprises (a) any of the anti-human OX40 agonist antibodies
described herein, (b) polysorbate 40, wherein the polysorbate
concentration is about 0.02% to about 0.06%; (c) a histidine
acetate buffer (e.g., a histidine acetate buffer at a pH between
5.0 and 6.0); and sucrose, wherein the sucrose concentration is
about 120 mM to about 320 mM.
[0444] In some embodiments, the pharmaceutical formulation
comprises (a) any of the anti-human OX40 agonist antibodies
described herein, (b) polysorbate 20, wherein the polysorbate
concentration is about 0.02%; (c) a histidine acetate buffer at pH
6.0; and (d) sucrose, wherein the sucrose concentration is about
320 mM.
[0445] In some embodiments, the pharmaceutical formulation
comprises (a) any of the anti-human OX40 agonist antibodies
described herein, (b) polysorbate 20, wherein the polysorbate
concentration is about 0.02%; (c) a histidine acetate buffer at pH
5.5; and (d) sucrose, wherein the sucrose concentration is about
240 mM.
[0446] In some embodiments, the pharmaceutical formulation
comprises (a) any of the anti-human OX40 agonist antibodies
described herein, (b) polysorbate 20, wherein the polysorbate
concentration is about 0.04%; (c) a histidine acetate buffer at pH
6.0; and (d) sucrose, wherein the sucrose concentration is about
120 mM.
[0447] In some embodiments, the pharmaceutical formulation
comprises (a) any of the anti-human OX40 agonist antibodies
described herein, (b) polysorbate 40, wherein the polysorbate
concentration is about 0.04%; (c) a histidine acetate buffer at pH
5.0; and (d) sucrose, wherein the sucrose concentration is about
240 mM.
[0448] In some embodiments, the pharmaceutical formulation
comprises (a) any of the anti-human OX40 agonist antibodies
described herein, (b) polysorbate 40, wherein the polysorbate
concentration is about 0.04%; (c) a histidine acetate buffer at pH
6.0; and (d) sucrose, wherein the sucrose concentration is about
120 mM.
[0449] In some embodiments, the pharmaceutical formulation is a
liquid pharmaceutical formulation. In some embodiments, the
pharmaceutical formulation is a stable pharmaceutical formulation.
In some embodiments, the pharmaceutical formulation is a stable
liquid pharmaceutical formulation.
[0450] In some embodiments of any of the pharmaceutical
formulations described herein, the anti-human OX40 agonist antibody
of the pharmaceutical formulation is present at a concentration
between about 10 mg/mL and about 100 mg/mL. In some embodiments,
the concentration of the human OX40 agonist antibody is between
about any of 10 mg/mL to 50 mg/mL, 10 mg/mL to 75 mg/mL, 25 mg/mL
to 75 mg/mL, 50 mg/mL to 100 mg/mL, 50 mg/mL to 75 mg/mL, and/or 75
mg/mL to 100 mg/mL. In some embodiments, the concentration of the
human OX40 agonist antibody is greater than about any of 20 mg/mL,
30 mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL, or 100 mg/mL.
[0451] The pharmaceutical formulation preferably comprises a
polysorbate. The polysorbate is generally included in an amount
which reduces aggregate formation (such as that which occurs upon
shaking or shipping). Examples of polysorbate include, but are not
limited to, polysorbate 20 (polyoxyethylene (20) sorbitan
monolaurate), polysorbate 40 (polyoxyethylene (20) sorbitan
monopalmitate), polysorbate 60 (polyoxyethylene (20) sorbitan
monostearate), and/or polysorbate 80 (polyoxyethylene (20) sorbitan
monooleate). In some embodiments, the polysorbate is polysorbate 20
(polyoxyethylene (20) sorbitan monolaurate). In some embodiments of
any of the pharmaceutical formulations described herein, the
polysorbate concentration is sufficient to minimize aggregation
and/or maintain stability upon long term storage and/or during
administration (e.g., after dilution in an IV bag). In some
embodiments, the polysorbate concentration is about 0.005% w/v,
about 0.02% w/v, about 0.04% w/v and less than about 0.1% w/v. In
some embodiments, the polysorbate concentration is greater than
0.01% w/v and less than about 0.1% w/v. In some embodiments, the
polysorbate concentration is about any of 0.005% w/v, about 0.02%
w/v, 0.03% w/v, 0.04% w/v, or 0.05% w/v. In some embodiments, the
polysorbate is present at a concentration of about 0.04% w/v. In
some embodiments, the polysorbate is present at a concentration of
about 0.02% w/v.
[0452] The pharmaceutical formulation preferably comprises a
saccharide. Saccharides include monosaccharides, disaccharides,
trisaccharides, polysaccharides, sugar alcohols, reducing sugars,
nonreducing sugars, etc. Further examples of saccharides include,
but are not limited to, glucose, sucrose, trehalose, lactose,
fructose, maltose, dextran, glycerin, dextran, erythritol,
glycerol, arabitol, sylitol, sorbitol, mannitol, mellibiose,
melezitose, raffinose, mannotriose, stachyose, maltose, lactulose,
maltulose, glucitol, maltitol, lactitol, iso-maltulose, etc. In
some embodiments, the saccharide is a disaccharide. In some
embodiments, the saccharide is a nonreducing disaccharide. In some
embodiments, the saccharide is trehalose.
[0453] The saccharide is generally included in an amount which
reduces aggregate formation. In some embodiments of any of the
pharmaceutical formulations described herein, the saccharide is
present at a concentration of between about any of 50 mM to 250 mM,
75 mM to 200 mM, 75 mM to 150 mM, 100 mM to 150 mM, or 110 mM to
130 mM, or 100 mM to 320 mM, or 240 mM to 320 mM, or 240 mM to 400
mM. In some embodiments, the saccharide is present at a
concentration greater than about any of 50 mM, 75 mM, 100 mM, 110
mM, or 115 mM. In some embodiments, the saccharide is present at a
concentration of about any of 100 mM, 110 mM, 120 mM, 130 mM, or
140 mM. In some embodiments, the saccharide is present at a
concentration of about 120 mM. In some embodiments of any of the
formulations, the saccharide is present at a concentration of about
75 mM to about 360 mM (e.g., about 100 mM, about 120 mM, about 240
mM, about 320 mM to about 360 mM). In some embodiments, the
saccharide is present at a concentration of about 240 mM. In some
embodiments, the saccharide is present at a concentration of about
320 mM.
[0454] The pharmaceutical formulation preferably comprises a
histidine buffer. Examples of histidine buffers include, but are
not limited to, histidine chloride, histidine succinate, histidine
acetate, histidine phosphate, histidine sulfate. In some
embodiments, the histidine buffer is histidine acetate. In some
embodiments of any of the pharmaceutical formulations described
herein, the histidine buffer concentration is between about any of
1 mM to 50 mM, 1 mM to 35 mM, 1 mM to 25 mM, 1 mM to 20 mM, 7.5 mM
to 12.5 mM, or 5 mM to 15 mM, 20 mM to 30 mM, 25 mM to 35 mM. In
some embodiments, the histidine buffer concentration is about any
of 5 mM, 7.5 mM, 10 mM, 12.5 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM
or 40 mM. In some embodiments, the histidine buffer concentration
is about 10 mM. In some embodiments, the histidine buffer
concentration is about 20 mM. In some embodiments, the histidine
buffer concentration is about 30 mM. In some embodiments, the
histidine buffer concentration is about 40 mM. In some embodiments
of any of the pharmaceutical formulations described herein, the
histidine buffer is at a pH of between pH 5.0 and 6.0, for example,
about any of pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH
5.6, pH 5.7, pH 5.8, pH 5.9 or pH 6.0. In some embodiments, the pH
is between pH 4.9 to pH 6.3.
[0455] The pharmaceutical formulation herein may also contain more
than one active compound as necessary for the particular indication
being treated, preferably those with complementary activities that
do not adversely affect each other. Such molecules are suitably
present in combination in amounts that are effective for the
purpose intended.
[0456] Further, provided herein are vials and methods of filing a
vial comprising a pharmaceutical formulation described herein. In
some embodiments, the pharmaceutical formulation is provided inside
a vial with a stopper pierceable by a syringe, preferably in
aqueous form. The vial is desirably stored at about 2-8.degree. C.
as well as up to 30.degree. C. for 24 hours until it is
administered to a subject in need thereof. The vial may for example
be a 15 cc vial (for example for a 200 mg dose).
[0457] The pharmaceutical formulation for administration is
preferably a liquid formulation (not lyophilized) and has not been
subjected to prior lyophilization. While the pharmaceutical
formulation may be lyophilized, preferably it is not. In some
embodiments of any of the pharmaceutical formulations, the
pharmaceutical formulation, the pharmaceutical formulation is a
lyophilized pharmaceutical formulation. In some embodiments, the
pharmaceutical formulation is a liquid formulation. In some
embodiments, the pharmaceutical formulation does not contain a
tonicifying amount of a salt such as sodium chloride. In some
embodiments of any of the pharmaceutical formulations, the
pharmaceutical formulation is diluted.
G. Therapeutic Methods and Compositions
[0458] Any of the anti-human OX40 antibodies and anti-PDL1
antibodies provided herein may be used in therapeutic methods. For
example, in certain aspects, the invention provides methods of
treating or delaying progression of cancer in an individual by
administering to the individual a dose of an anti-human OX40
agonist antibody of the present disclosure and a dose of an
anti-PDL1 antibody of the present disclosure. In some embodiments,
the dose(s) of the anti-human OX40 agonist antibody and/or
anti-PDL1 antibody of the present disclosure may be part of a
pharmaceutical formulation. In some embodiments, the anti-human
OX40 agonist antibody comprises (a) HVR-H1 comprising the amino
acid sequence of SEQ ID NO:2; (b) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:3; (c) HVR-H3 comprising the amino acid
sequence of SEQ ID NO:4; (d) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:6; and (f) HVR-L3 comprising an amino acid
sequence selected from SEQ ID NO:7. In certain embodiments, the
anti-human OX40 agonist antibody is MOXR0916 (1A7.gr1 IgG1). In
some embodiments, the anti-PDL1 antibody comprises (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO:196; (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO:197; (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:198; (d) HVR-L1
comprising the amino acid sequence of SEQ ID NO:199; (e) HVR-L2
comprising the amino acid sequence of SEQ ID NO:200; and (f) HVR-L3
comprising an amino acid sequence selected from SEQ ID NO:201. In
certain embodiments, the anti-PDL1 antibody is MPDL3280A.
[0459] In some embodiments, the dose may be between about 0.5 mg
and about 1500 mg of the anti-human OX40 agonist antibody. For
example, the dose of the anti-human OX40 agonist antibody may be
between about 0.5 mg and about 1500 mg, between about 0.5 mg and
about 1400 mg, between about 0.5 mg and about 1200 mg, between
about 0.5 mg and about 1000 mg, between about 0.5 mg and about 800
mg, between about 0.5 mg and about 600 mg, between about 0.5 mg and
about 500 mg, between about 0.5 mg and about 400 mg, between about
0.5 mg and about 200 mg, between about 0.5 mg and about 150 mg,
between about 0.5 mg and about 100 mg, between about 0.5 mg and
about 50 mg, between about 0.5 mg and about 25 mg, between about
0.5 mg and about 15 mg, between about 0.5 mg and about 10 mg,
between about 0.5 mg and about 5 mg, or between about 0.5 mg and
about 1 mg. In some embodiments, the dose is less than about any of
the following doses (in mg): 1500, 1400, 1200, 1000, 800, 600, 500,
400, 200, 150, 100, 50, 25, 15, 10, 5, or 1. In some embodiments,
the dose is greater than about any of the following doses (in mg):
0.5, 0.8, 1, 5, 10, 15, 25, 50, 100, 150, 200, 400, 500, 600, 800,
1000, 1200, or 1400. That is, the dose can be any of a range of
doses (in mg) having an upper limit of 1500, 1400, 1200, 1000, 800,
600, 500, 400, 200, 150, 100, 50, 25, 15, 10, 5, or 1 and an
independently selected lower limit of 0.5, 0.8, 1, 5, 10, 15, 25,
50, 100, 150, 200, 400, 500, 600, 800, 1000, 1200, or 1400, wherein
the lower limit is less than the upper limit.
[0460] In some embodiments, the anti-human OX40 agonist antibody
dose is selected from about 0.8 mg, about 3.2 mg, about 12 mg,
about 40 mg, about 80 mg, about 130 mg, about 160 mg, about 300 mg,
about 320 mg, about 400 mg, about 600 mg, and about 1200 mg, e.g.,
per administration. In certain embodiments, the anti-human OX40
agonist antibody dose is about 300 mg. In certain embodiments, the
anti-human OX40 agonist antibody dose is selected from 0.8 mg, 3.2
mg, 12 mg, 40 mg, 80 mg, 130 mg, 160 mg, 300 mg, 320 mg, 400 mg,
600 mg, and 1200 mg. In certain embodiments, the anti-human OX40
agonist antibody dose is 300 mg.
[0461] In some embodiments, the anti-human OX40 agonist antibody
dose is selected from about 0.5 mg, about 2 mg, about 8 mg, about
27 mg, about 53 mg, about 87 mg, about 107 mg, about 200 mg, about
213 mg, about 267 mg, about 400 mg, and about 800 mg, e.g., per
administration. In certain embodiments, the anti-human OX40 agonist
antibody dose is selected from 0.5 mg, 2 mg, 8 mg, 27 mg, 53 mg, 87
mg, 107 mg, 200 mg, 213 mg, 267 mg, 400 mg, and 800 mg.
[0462] In some embodiments, the administration of the anti-human
OX40 agonist antibody may be repeated at one or more additional
doses. In some embodiments, each dose of the one or more additional
doses is selected from about 0.8 mg, about 3.2 mg, about 12 mg,
about 40 mg, about 80 mg, about 130 mg, about 160 mg, about 300 mg,
about 320 mg, about 400 mg, about 600 mg, and about 1200 mg, e.g.,
per administration. In some embodiments, each dose of the one or
more additional doses is about 300 mg.
[0463] The administration of the anti-human OX40 agonist antibody
may be adjusted, e.g., based on the dosing cycle. In some
embodiments, the anti-human OX40 agonist antibody dose is selected
from about 0.8 mg, about 3.2 mg, about 12 mg, about 40 mg, about 80
mg, about 130 mg, about 160 mg, about 300 mg, about 320 mg, about
400 mg, about 600 mg, and about 1200 mg, e.g., per administration,
and the anti-human OX40 agonist antibody may be administered at an
interval of about 3 weeks or about 21 days between each
administration. In some embodiments, the anti-human OX40 agonist
antibody dose is selected from about 0.5 mg, about 2 mg, about 8
mg, about 27 mg, about 53 mg, about 87 mg, about 107 mg, about 200
mg, about 213 mg, about 267 mg, about 400 mg, and about 800 mg,
e.g., per administration, and the anti-human OX40 agonist antibody
may be administered at an interval of about 2 weeks or about 14
days between each administration. In some embodiments, the dosing
interval for the anti-human OX40 agonist antibody may be adjusted,
e.g., to match a dosing interval or protocol of a concomitant
therapeutic agent or protocol (e.g., a 2-week dosing interval for
FOLFOX).
[0464] In some embodiments, 1-10 additional doses of the anti-human
OX40 agonist antibody are administered, e.g., in repeated
administration as described above. For example, in some
embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional doses of
the anti-human OX40 agonist antibody may be administered.
[0465] In some embodiments, each dose of the anti-human OX40
agonist antibody administered to the individual may be the same. In
other embodiments, each dose of the anti-human OX40 agonist
antibody administered to the individual is not the same. Dosing may
be modified as described herein, e.g., based on efficacy, toxicity,
adverse events, progression, PD, PK, an effect of a second
therapeutic agent (e.g., an anti-PDL1 antibody), and so forth.
[0466] Any effective dose known in the art for an anti-PDL1
antibody may be used. In some embodiments, the anti-PDL1 antibody
is administered at a dose of about 800 mg or about 1200 mg. The
administration of the anti-PDL1 may be adjusted, e.g., based on the
dosing cycle. For example, in certain embodiments, the anti-PDL1
antibody is administered at a dose of 800 mg every 2 weeks.
Similarly, in certain embodiments, the anti-PDL1 antibody is
administered at a dose of 1200 mg every 3 weeks.
[0467] In some embodiments, 1-10 additional doses of the anti-PDL1
antibody are administered, e.g., in repeated administration as
described above. For example, in some embodiments, 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10 additional doses of the anti-PDL1 antibody may be
administered.
[0468] In some embodiments, the anti-human OX40 agonist antibody
and/or the anti-PDL1 antibody are administered intravenously. In
some embodiments, the anti-human OX40 agonist antibody and/or the
anti-PDL1 antibody may be administered at different rates between
administrations. For example, as described herein, an initial
administration may be performed at a slower rate (e.g., by IV
infusion) than a subsequent administration, e.g., to prevent or
mitigate infusion-related reactions.
[0469] In some embodiments, the anti-human OX40 agonist antibody
and the anti-PDL1 antibody are administered on the same day. In
other embodiments, the anti-human OX40 agonist antibody and the
anti-PDL1 antibody are administered on different days. In some
embodiments, the anti-human OX40 agonist antibody and the anti-PDL1
antibody are administered within 1 day, within 2 days, within 3
days, within 4 days, within 5 days, within 6 days, or within 7
days. In some embodiments, dosing may be staggered within a dosing
cycle, e.g., the anti-human OX40 agonist antibody may be
administered at each dosing interval (e.g., 2 or 3 weeks), and the
anti-PDL1 antibody may be administered every other dosing interval,
or vice versa.
[0470] In some embodiments, after administration of a first dose of
the anti-human OX40 agonist antibody and/or a first dose of the
anti-PDL1 antibody, one or more additional doses of the anti-human
OX40 agonist antibody and/or the anti-PDL1 antibody may be
administered. In some embodiments, after administering the
antibody, the individual is monitored for an adverse event (e.g.,
as exemplified below), progression and/or treatment efficacy. In
some embodiments, if the individual does not exhibit an adverse
event (e.g., as described herein), a second dose of the antibody
may be administered. In some embodiments, if the treatment exhibits
efficacy, a second dose of the antibody may be administered. In
some embodiments, even if progression is observed, a second dose of
the antibody may be administered. As described herein, and without
wishing to be bound to theory, it is thought that in some cases
immunotherapeutic agents such as anti-human OX40 agonist antibodies
and/or anti-PDL1 antibodies may induce an initial progression,
followed by a response.
[0471] In some embodiments, the second dose of the anti-human OX40
agonist antibody is the same amount as the first dose of the
anti-human OX40 agonist antibody. In other embodiments, the second
dose of the anti-human OX40 agonist antibody may be greater than
the first dose of the anti-human OX40 agonist antibody. In some
embodiments, the second dose of the anti-PDL1 antibody is the same
amount as the first dose of the anti-PDL1 antibody. It will be
appreciated that the particular doses and dose ranges of the
anti-human OX40 agonist antibody described above may apply to
second doses as well as first doses in any combination or
order.
[0472] In some embodiments, the second dose of the anti-human OX40
agonist antibody is not provided until from about 2 weeks to about
4 weeks after the first dose. In some embodiments, the second dose
of the anti-human OX40 agonist antibody is not provided until from
about 14 days, from about 21 days, or from about 28 days after the
first dose. In some embodiments, the second dose of the anti-PDL1
antibody is not provided until from about 2 weeks to about 4 weeks
after the first dose. In some embodiments, the second dose of the
anti-PDL1 antibody is not provided until about 14 days, about 21
days, or about 28 days after the first dose.
[0473] In some embodiments, the first dose of the anti-human OX40
agonist antibody and the first dose of the anti-PDL1 antibody are
administered on the same day. In other embodiments, the first dose
of the anti-human OX40 agonist antibody and the first dose of the
anti-PDL1 antibody are administered on the different days. In some
embodiments, the first dose of the anti-human OX40 agonist antibody
and the first dose of the anti-PDL1 antibody are administered
within 1 day, within 2 days, within 3 days, within 4 days, within 5
days, within 6 days, or within 7 days.
[0474] In some embodiments, the second dose of the anti-human OX40
agonist antibody and the second dose of the anti-PDL1 antibody are
administered on the same day. In other embodiments, the second dose
of the anti-human OX40 agonist antibody and the second dose of the
anti-PDL1 antibody are administered on the different days. In some
embodiments, the second dose of the anti-human OX40 agonist
antibody and the second dose of the anti-PDL1 antibody are
administered within 1 day, within 2 days, within 3 days, within 4
days, within 5 days, within 6 days, or within 7 days.
[0475] In some embodiments, the second dose of the anti-human OX40
agonist antibody and the second dose of the anti-PDL1 antibody are
not provided until about 3 weeks after the first dose of the
anti-human OX40 agonist antibody and the first dose of the
anti-human OX40 agonist antibody. In some embodiments, the second
dose of the anti-human OX40 agonist antibody and the second dose of
the anti-PDL1 antibody are not provided until about 21 days after
the first dose of the anti-human OX40 agonist antibody and the
first dose of the anti-human OX40 agonist antibody.
[0476] In some embodiments, the first dose and the second dose of
each antibody are administered via the same route. In certain
embodiments, the first dose of the anti-human OX40 agonist
antibody, the first dose of the anti-PDL1 antibody, the second dose
of the anti-human OX40 agonist antibody, and/or the second dose of
the anti-PDL1 antibody are administered intravenously.
[0477] In one aspect, an anti-human OX40 agonist antibody and an
anti-PDL1 antibody for use as a medicament are provided. In further
aspects, an anti-human OX40 agonist antibody and an anti-PDL1
antibody for use in treating cancer are provided. In certain
embodiments, an anti-human OX40 agonist antibody and an anti-PDL1
antibody for use in a method of treatment are provided. In certain
embodiments, the invention provides an anti-human OX40 agonist
antibody and an anti-PDL1 antibody for use in a method of treating
an individual having cancer comprising administering to the
individual an effective amount of the anti-human agonist OX40
antibody in conjunction with an effective amount of an anti-PDL1
antibody. In one such embodiment, the method further comprises
administering to the individual an effective amount of at least one
additional therapeutic agent, e.g., as described below.
[0478] In one aspect, provided is an anti-human OX40 agonist
antibody for use in enhancing immune function (e.g., by
upregulating cell-mediated immune responses) in an individual
having cancer comprising administering to the individual an
effective amount of the anti-human agonist OX40 antibody. In one
aspect, provided is an anti-human OX40 agonist antibody for use in
enhancing T cell function in an individual having cancer comprising
administering to the individual an effective amount of the
anti-human agonist OX40 antibody. In one aspect, provided are an
anti-human OX40 agonist antibody for use in depleting human
OX40-expressing cells (e.g., OX40 expressing T cells, e.g., OX40
expressing Treg) comprising administering to the individual an
effective amount of the anti-human agonist OX40 antibody. In some
embodiments, depletion is by ADCC. In some embodiments, depletion
is by phagocytosis. Provided is an anti-human OX40 agonist antibody
for treating an individual having tumor immunity.
[0479] In further aspects, an anti-human OX40 agonist antibody for
use in treating infection (e.g., with a bacteria or virus or other
pathogen) is provided. In certain embodiments, the invention
provides an anti-human OX40 agonist antibody for use in a method of
treating an individual having an infection comprising administering
to the individual an effective amount of the anti-human agonist
OX40 antibody. In some embodiments, the infection is with a virus
and/or a bacteria. In some embodiments, the infection is with a
pathogen.
[0480] In a further aspect, the invention provides for the use of
an anti-OX40 antibody in the manufacture or preparation of a
medicament. In a further aspect, the invention provides for the use
of an anti-PDL1 antibody in the manufacture or preparation of a
medicament. In one embodiment, the medicament is for treatment of
cancer. In a further embodiment, the medicament is for use in a
method of treating cancer comprising administering to an individual
having cancer an effective amount of the medicament containing
anti-OX40 antibody and the medicament containing anti-PDL1
antibody. In one such embodiment, the method further comprises
administering to the individual an effective amount of at least one
additional therapeutic agent, e.g., as described below.
[0481] In one aspect, the medicament is for use in enhancing immune
function (e.g., by upregulating cell-mediated immune responses) in
an individual having cancer comprising administering to the
individual an effective amount of the medicament. In one aspect,
the medicament is for use in enhancing T cell function in an
individual having cancer comprising administering to the individual
an effective amount of the medicament. In some embodiments, the T
cell dysfunctional disorder is cancer. In one aspect, the
medicament is for use in depleting human OX40-expressing cells
(e.g., cell expressing high OX40, e.g., OX40 expressing T cells)
comprising administering to the individual an effective amount of
the medicament. In some embodiments, depletion is by ADCC. In some
embodiments, depletion is by phagocytosis. In one aspect, the
medicament is for treating an individual having tumor immunity.
[0482] In further aspects, the medicament is for use in treating
infection (e.g., with a bacteria or virus or other pathogen) is
provided. In certain embodiments, the medicament is for use in a
method of treating an individual having an infection comprising
administering to the individual an effective amount of the
medicament. In some embodiments, the infection is with virus and/or
bacteria. In some embodiments, the infection is with a
pathogen.
[0483] In a further aspect, the invention provides a method for
treating a cancer. In one embodiment, the method comprises
administering to an individual having such cancer an effective
amount of an anti-OX40 antibody and an effective amount of an
anti-PDL1 antibody. In one such embodiment, the method further
comprises administering to the individual an effective amount of at
least one additional therapeutic agent, as described below. An
"individual" according to any of the above embodiments may be a
human.
[0484] In one aspect, provided is a method for enhancing immune
function (e.g., by upregulating cell-mediated immune responses) in
an individual having cancer comprising administering to the
individual an effective amount of the anti-human agonist OX40
antibody and an effective amount of an anti-PDL1 antibody. In one
aspect, provided is a method for enhancing T cell function in an
individual having cancer comprising administering to the individual
an effective amount of the anti-human agonist OX40 antibody and an
effective amount of an anti-PDL1 antibody. In one aspect, provided
are a method for depleting human OX40-expressing cells (e.g., cells
that express high level of OX40, e.g., OX40 expressing T cells)
comprising administering to the individual an effective amount of
the anti-human agonist OX40 antibody. In some embodiments,
depletion is by ADCC. In some embodiments, depletion is by
phagocytosis. Provided are an anti-human OX40 agonist antibody and
an effective amount of an anti-PDL1 antibody for treating an
individual having tumor immunity.
[0485] In some embodiments, examples of cancer further include, but
are not limited to, B-cell lymphoma (including low grade/follicular
non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL;
intermediate grade/follicular NHL; intermediate grade diffuse NHL;
high grade immunoblastic NHL; high grade lymphoblastic NHL; high
grade small non-cleaved cell NHL; bulky disease NHL; mantle cell
lymphoma; AIDS-related lymphoma; and Waldenstrom's
Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute
lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic
myeloblastic leukemia; and post-transplant lymphoproliferative
disorder (PTLD), as well as abnormal vascular proliferation
associated with phakomatoses, edema (such as that associated with
brain tumors), B-cell proliferative disorders, and Meigs' syndrome.
More specific examples include, but are not limited to, relapsed or
refractory NHL, front line low grade NHL, Stage III/IV NHL,
chemotherapy resistant NHL, precursor B lymphoblastic leukemia
and/or lymphoma, small lymphocytic lymphoma, B-cell chronic
lymphocytic leukemia and/or prolymphocytic leukemia and/or small
lymphocytic lymphoma, B-cell prolymphocytic lymphoma, immunocytoma
and/or lymphoplasmacytic lymphoma, lymphoplasmacytic lymphoma,
marginal zone B-cell lymphoma, splenic marginal zone lymphoma,
extranodal marginal zone--MALT lymphoma, nodal marginal zone
lymphoma, hairy cell leukemia, plasmacytoma and/or plasma cell
myeloma, low grade/follicular lymphoma, intermediate
grade/follicular NHL, mantle cell lymphoma, follicle center
lymphoma (follicular), intermediate grade diffuse NHL, diffuse
large B-cell lymphoma, aggressive NHL (including aggressive
front-line NHL and aggressive relapsed NHL), NHL relapsing after or
refractory to autologous stem cell transplantation, primary
mediastinal large B-cell lymphoma, primary effusion lymphoma, high
grade immunoblastic NHL, high grade lymphoblastic NHL, high grade
small non-cleaved cell NHL, bulky disease NHL, Burkitt's lymphoma,
precursor (peripheral) large granular lymphocytic leukemia, mycosis
fungoides and/or Sezary syndrome, skin (cutaneous) lymphomas,
anaplastic large cell lymphoma, angiocentric lymphoma.
[0486] In some embodiments, examples of cancer further include, but
are not limited to, B-cell proliferative disorders, which further
include, but are not limited to, lymphomas (e.g., B-Cell
Non-Hodgkin's lymphomas (NHL)) and lymphocytic leukemias. Such
lymphomas and lymphocytic leukemias include e.g. a) follicular
lymphomas, b) Small Non-Cleaved Cell Lymphomas/Burkitt's lymphoma
(including endemic Burkitt's lymphoma, sporadic Burkitt's lymphoma
and Non-Burkitt's lymphoma), c) marginal zone lymphomas (including
extranodal marginal zone B-cell lymphoma (Mucosa-associated
lymphatic tissue lymphomas, MALT), nodal marginal zone B-cell
lymphoma and splenic marginal zone lymphoma), d) Mantle cell
lymphoma (MCL), e) Large Cell Lymphoma (including B-cell diffuse
large cell lymphoma (DLCL), Diffuse Mixed Cell Lymphoma,
Immunoblastic Lymphoma, Primary Mediastinal B-Cell Lymphoma,
Angiocentric Lymphoma-Pulmonary B-Cell Lymphoma), f) hairy cell
leukemia, g) lymphocytic lymphoma, Waldenstrom's macroglobulinemia,
h) acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia
(CLL)/small lymphocytic lymphoma (SLL), B cell prolymphocytic
leukemia, i) plasma cell neoplasms, plasma cell myeloma, multiple
myeloma, plasmacytoma, and/or j) Hodgkin's disease.
[0487] In some embodiments of any of the methods, the cancer is
melanoma, triple-negative breast cancer, ovarian cancer, renal cell
cancer, bladder cancer, non-small cell lung cancer, gastric cancer,
or colorectal cancer (including both primary and metastatic
tumors). In certain embodiments, the cancer is a renal cell
carcinoma (e.g., clear cell renal cell carcinoma).
[0488] In some embodiments of any of the methods, the cancer is a
B-cell proliferative disorder. In some embodiments, the B-cell
proliferative disorder is lymphoma, non-Hodgkins lymphoma (NHL),
aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL,
refractory NHL, refractory indolent NHL, chronic lymphocytic
leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell
leukemia (HCL), acute lymphocytic leukemia (ALL), or mantle cell
lymphoma. In some embodiments, the B-cell proliferative disorder is
NHL, such as indolent NHL and/or aggressive NHL. In some
embodiments, the B-cell proliferative disorder is indolent
follicular lymphoma or diffuse large B-cell lymphoma. In certain
embodiments, the cancer is selected from melanoma, triple-negative
breast cancer, ovarian cancer, renal cell cancer, bladder cancer,
non-small cell lung cancer, gastric cancer, and colorectal cancer.
In some embodiments, the cancer is a locally advanced or metastatic
solid tumor, e.g., of any of the solid cancers described
herein.
[0489] In some embodiments, the cancer is melanoma. In certain
embodiments, the melanoma is advanced or metastatic melanoma. In
some embodiments, the melanoma exhibits a BRAF V600 mutation (e.g.,
a V600E, V600K, or V600D mutation). Melanomas with a BRAF V600
mutation have been treated with B-Raf and/or mitogen-activated
protein kinase kinase (MEK) kinase inhibitors. Examples of such
inhibitors include without limitation sorafenib, vemurafenib,
dabrafenib (GSK2118436), RAF265, LGX818, trametinib, selumetinib,
binimetinib, cobimetinib, PD-325901, CI-1040 (PD184352), PD035901,
and the like. In some embodiments, the individual has been treated
with a B-Raf and/or mitogen-activated protein kinase kinase (MEK)
kinase inhibitor prior to treatment with the anti-human OX40
agonist antibody and/or anti-PDL1 antibody. In some embodiments,
the patient has exhibited disease progression or intolerance to the
B-Raf and/or mitogen-activated protein kinase kinase (MEK) kinase
inhibitor treatment prior to treatment with the anti-human OX40
agonist antibody and/or anti-PDL1 antibody.
[0490] In some embodiments, the cancer is renal cell cancer (RCC).
In certain embodiments, the RCC is advanced or metastatic RCC. In
some embodiments, the RCC exhibits a component of clear cell
histology and/or a component of sarcomatoid histology.
[0491] In some embodiments, the cancer is triple-negative breast
cancer (TNBC). In certain embodiments, the TNBC is advanced or
metastatic TNBC. In some embodiments, TNBC may refer to an
adenocarcinoma of the breast that is estrogen receptor negative,
progesterone receptor negative, and human epidermal growth factor
receptor 2 negative, e.g., as defined by the American Society of
Clinical Oncology-College of American Pathologists (ASCO-CAP)
guidelines. For example, <1% of tumor cell nuclei may be
immunoreactive for estrogen receptor, and <1% of tumor cell
nuclei may be immunoreactive for progesterone receptor (Hammond, M.
E. et al. (2010) J. Clin. Oncol. 28:2784-2795) and HER2 tests
demonstrate either immunohistochemistry (IHC) 1+, IHC 0 or in situ
hybridization (ISH) negative (Wolff, A. C. et al. (2013) J. Clin.
Oncol. 31:3997:4013).
[0492] In some embodiments, the cancer is non-small cell lung
cancer (NSCLC). In certain embodiments, the NSCLC is advanced or
metastatic NSCLC. In some embodiments, the NSCLC exhibits a
sensitizing epidermal growth factor (EGFR) mutation. Sensitizing
EGFR mutations are known to involve the EGFR kinase domain and may
include without limitation mutations in exons 18-21, such as exon
19 deletions and the L858R point mutation in exon 21 (for further
description and/or additional mutations, see, e.g., Lynch, T. J. et
al. (2004) N. Engl. J. Med. 350:2129-2139; Pao, W. et al. (2004)
Proc. Natl. Acad. Sci. 101:13306-13311; and Paez, J. G. et al.
(2004) Science 304:1497-1500). In some embodiments, the individual
has been treated with an EGFR tyrosine kinase inhibitor prior to
treatment with the anti-human OX40 agonist antibody and/or
anti-PDL1 antibody. In some embodiments, the patient has exhibited
disease progression or intolerance to the EGFR tyrosine kinase
inhibitor treatment prior to treatment with the anti-human OX40
agonist antibody and/or anti-PDL1 antibody. In some embodiments,
the NSCLC exhibits an anaplastic lymphoma kinase (ALK)
rearrangement. ALK rearrangements have been implicated in NSCLC,
particularly in EGFR tyrosine kinase inhibitor resistance, and many
ALK rearrangements are known in the art, including without
limitation EML4-ALK, KIF5B-ALK, and TFG-ALK rearrangements (for
further description and/or additional mutations, see, e.g.,
Koivunen, J. P. et al. (2008) Clin. Cancer Res. 14:4275-4283; and
Soda, E. M. et al. (2007) Nature 448:561-566). In some embodiments,
the individual has been treated with an ALK tyrosine kinase
inhibitor prior to treatment with the anti-human OX40 agonist
antibody and/or anti-PDL1 antibody. In some embodiments, the
patient has exhibited disease progression or intolerance to the ALK
tyrosine kinase inhibitor treatment prior to treatment with the
anti-human OX40 agonist antibody and/or anti-PDL1 antibody.
[0493] In some embodiments, the cancer is urothelial bladder cancer
(UBC). In certain embodiments, the UBC is advanced or metastatic
UBC. In some embodiments, the UBC exhibits a transitional cell
pattern and includes carcinomas of the renal pelvis, ureters,
urinary bladder, and/or urethra.
[0494] In some embodiments, the cancer is colorectal cancer (CRC).
In certain embodiments, the CRC is advanced or metastatic CRC. In
some embodiments, the CRC is an adenocarcinoma of the colon or
rectum. In some embodiments, the CRC exhibits microsatellite
instability-high (MSI-H) status. Approximately 15% of colorectal
cancers demonstrate deficiencies in the DNA mismatch repair system
(Boland et al. (1998) Cancer Res. 58:5248-5257). These deficiencies
are predominantly nonfamilial (sporadic) and lead to an
accumulation of somatic mutations particularly in repetitive
sequences (mono-, di-, or higher-order nucleotide repeats) and
microsatellites. Hence, a defining molecular feature of these
tumors is a high level of microsatellite instability, or MSI-H. The
associated insertions or deletions in repetitive sequences
occurring in coding regions of the genome can lead to the
expression and display of mutant peptides, some of which are
capable of eliciting T-cell responses (Bauer et al. (2013) Cancer
Immunol. Immunother. 62:27-37). The MSI-H phenotype is also
associated with mutations in specific oncogenes and tumor
suppressors including BRAF and MRE11A (Vilar and Gruber (2010) Nat.
Rev. Clin. Oncol. 7:153-162). Without wishing to be bound to
theory, MSI-H tumors may therefore exhibit higher immunogenicity in
comparison to microsatellite-stable tumors. One possible correlate
of higher immunogenicity is that MSI-H CRC is characterized by the
presence of high numbers of tumor-infiltrating lymphocytes
(Greenson et al. (2003) Am. J. Surg. Pathol. 27:563-570).
[0495] In some embodiments, the cancer is ovarian cancer (OC). In
certain embodiments, the OC is advanced or metastatic OC. In some
embodiments, the OC is an epithelial ovarian, fallopian tube, or
primary peritoneal cancer.
[0496] In some embodiments of any of the methods, the tumor or
cancer is refractory. As used herein, the term "refractory" may
refer to a tumor/cancer, or be used to describe a patient with said
tumor/cancer, for which a prior therapy has been ineffective and/or
intolerable. For example, for RCC, a "refractory" patient may be
one for whom prior anti-cancer therapy comprising a VEGF inhibitor
and/or an mTOR inhibitor has proven to be ineffective and/or
intolerable. One skilled in the art will appreciate that such
therapies are merely exemplary, and the methods of the present
disclosure may be used to treat or delay progression of a cancer
such as RCC or any of the other cancers described herein that is
refractory to one or more other therapies, as the appropriateness
of the benefit/risk profile of an anti-cancer therapy may in some
cases be up to clinical judgement of the prescribing
oncologist.
[0497] In some embodiments of any of the methods, the individual
has been previously treated with an immunotherapy agent prior to
the administration of the anti-human OX40 agonist antibody and the
anti-PDL1 antibody (and optionally a VEGF antagonist such as
bevacizumab). It is a surprising finding of the clinical trials
disclosed herein that treatment with anti-human OX40 agonist
antibody and anti-PDL1 antibody may be efficacious (e.g., resulting
in immune activation and/or PD modulation) even in patients
previously treated with anti-human OX40 agonist antibody or PD-1
axis binding antagonist as a single-agent or monotherapy treatment.
A variety of immunotherapy agents are described herein (the term
"immunotherapeutic agent" may be used interchangeably herein). In
certain embodiments, the immunotherapy agent is an anti-human OX40
agonist antibody. In certain embodiments, the immunotherapy agent
is a PD-1 axis binding antagonist (e.g., an anti-PDL1, anti-PDL2,
or anti-PD1 antibody). In some embodiments, the immunotherapy agent
is an OX40 agonist, such as an anti-human OX40 agonist antibody. In
some embodiments, the prior treatment with the immunotherapy agent
is a monotherapy or single-agent treatment. For example, in some
embodiments, the prior treatment with the immunotherapy agent
comprises treatment with an OX40 agonist (e.g., an anti-human OX40
agonist antibody) in the absence of a PD-1 axis binding antagonist
(e.g., an anti-PDL1, anti-PDL2, or anti-PD1 antibody). In other
embodiments, the prior treatment with the immunotherapy agent
comprises treatment with a PD-1 axis binding antagonist (e.g., an
anti-PDL1, anti-PDL2, or anti-PD1 antibody) in the absence of an
OX40 agonist (e.g., an anti-human OX40 agonist antibody). In some
embodiments, the individual exhibited a stable disease response,
disease progression, and/or intolerance to a prior treatment prior
to the administration of the anti-human OX40 agonist antibody and
the anti-PDL1 antibody (and optionally aVEGF antagonist such as
bevacizumab).
[0498] In some embodiments, provided herein is a method of treating
or delaying progression of cancer in an individual comprising
administering to the individual (i) MOXR0916 at a dose of 300 mg,
and (ii) atezolizumab at a dose of 1200 mg, wherein the cancer is
selected from the group consisting of melanoma, triple-negative
breast cancer, ovarian cancer, renal cell cancer, bladder cancer,
non-small cell lung cancer, gastric cancer, and colorectal cancer.
In certain embodiments, the cancer is renal cell cancer. In certain
embodiments, the cancer is colorectal cancer. In some embodiments,
the MOXR0916 and atezolizumab are administered on the same day. In
some embodiments, the method further comprises repeating the
administration of MOXR0916 at a dose of 300 mg per administration
and atezolizumab at a dose of 1200 mg per administration, and
wherein the administration is repeated at an interval of about 3
weeks or about 21 days between administrations. In some
embodiments, the repeated administrations of the MOXR0916 and the
atezolizumab are administered on the same day. In some embodiments,
the cancer is RCC. In some embodiments, the cancer is bladder
cancer. In some embodiments, MOXR0916 is administered
intravenously. In some embodiments, atezolizumab is administered
intravenously. In some embodiments, MOXR0916 and atezolizumab are
administered intravenously.
[0499] In some embodiments, provided herein is a method of treating
or delaying progression of cancer in an individual comprising
administering to the individual (i) MOXR0916 at a dose of 160 mg,
and (ii) atezolizumab at a dose of 1200 mg, wherein the cancer is
selected from the group consisting of melanoma, triple-negative
breast cancer, ovarian cancer, renal cell cancer, bladder cancer,
non-small cell lung cancer, gastric cancer, and colorectal cancer.
In certain embodiments, the cancer is renal cell cancer. In certain
embodiments, the cancer is colorectal cancer. In some embodiments,
the MOXR0916 and the atezolizumab are administered on the same day.
In some embodiments, the method further comprises repeating the
administration of MOXR0916 at a dose of 160 mg per administration
and atezolizumab at a dose of 1200 mg per administration, and
wherein the administration is repeated at an interval of about 3
weeks or about 21 days between administrations. In some
embodiments, the repeated administrations of the MOXR0916 and the
atezolizumab are administered on the same day. In some embodiments,
the cancer is RCC. In some embodiments, the cancer is bladder
cancer. In some embodiments, MOXR0916 is administered
intravenously. In some embodiments, atezolizumab is administered
intravenously. In some embodiments, MOXR0916 and atezolizumab are
administered intravenously.
[0500] In some embodiments, provided herein is a method of treating
or delaying progression of cancer in an individual comprising
administering to the individual (i) MOXR0916 at a dose of 320 mg,
and (ii) atezolizumab at a dose of 1200 mg, wherein the cancer is
selected from the group consisting of melanoma, triple-negative
breast cancer, ovarian cancer, renal cell cancer, bladder cancer,
non-small cell lung cancer, gastric cancer, and colorectal cancer.
In certain embodiments, the cancer is renal cell cancer. In certain
embodiments, the cancer is colorectal cancer. In some embodiments,
the MOXR0916 and atezolizumab are administered on the same day. In
some embodiments, the method further comprises repeating the
administration of MOXR0916 at a dose of 320 mg per administration
and atezolizumab at a dose of 1200 mg per administration, and
wherein the administration is repeated at an interval of about 3
weeks or about 21 days between administrations. In some
embodiments, the repeated administrations of the MOXR0916 and the
atezolizumab are administered on the same day. In some embodiments,
the cancer is RCC. In some embodiments, the cancer is bladder
cancer. In some embodiments, MOXR0916 is administered
intravenously. In some embodiments, atezolizumab is administered
intravenously. In some embodiments, MOXR0916 and atezolizumab are
administered intravenously.
[0501] In some embodiments, provided herein is a method of treating
or delaying progression of cancer in an individual comprising
administering to the individual (i) MOXR0916 at a dose of 400 mg,
and (ii) atezolizumab at a dose of 1200 mg, wherein the cancer is
selected from the group consisting of melanoma, triple-negative
breast cancer, ovarian cancer, renal cell cancer, bladder cancer,
non-small cell lung cancer, gastric cancer, and colorectal cancer.
In certain embodiments, the cancer is renal cell cancer. In certain
embodiments, the cancer is colorectal cancer. In some embodiments,
the MOXR0916 and atezolizumab are administered on the same day. In
some embodiments, the method further comprises repeating the
administration of MOXR0916 at a dose of 400 mg per administration
and atezolizumab at a dose of 1200 mg per administration, and
wherein the administration is repeated at an interval of about 3
weeks or about 21 days between administrations. In some
embodiments, the repeated administrations of the MOXR0916 and the
atezolizumab are administered on the same day. In some embodiments,
the cancer is RCC. In some embodiments, the cancer is bladder
cancer. In some embodiments, MOXR0916 is administered
intravenously. In some embodiments, atezolizumab is administered
intravenously. In some embodiments, MOXR0916 and atezolizumab are
administered intravenously.
[0502] In some embodiments, provided herein is a method of treating
or delaying progression of cancer in an individual comprising
administering to the individual (i) MOXR0916 at a dose of 300 mg,
(ii) atezolizumab at a dose of 1200 mg, and (iii) bevacizumab at a
dose of 15 mg/kg, wherein the cancer is selected from the group
consisting of melanoma, triple-negative breast cancer, ovarian
cancer, renal cell cancer, bladder cancer, non-small cell lung
cancer, gastric cancer, and colorectal cancer. In certain
embodiments, the cancer is renal cell cancer. In certain
embodiments, the cancer is colorectal cancer. In some embodiments,
the MOXR0916, atezolizumab, and bevacizumab are administered on the
same day. In some embodiments, the method further comprises
repeating the administration of MOXR0916 at a dose of 300 mg per
administration, atezolizumab at a dose of 1200 mg per
administration, and bevacizumab at a dose of 15 mg/kg per
administration. In some embodiments, the administration of
MOXR0916, atezolizumab, and bevacizumab is repeated at an interval
of about 3 weeks or about 21 days between administrations. In some
embodiments, the repeated administrations of the MOXR0916, the
atezolizumab, and the bevacizumab are administered on the same day.
In some embodiments, MOXR0916 is administered intravenously. In
some embodiments, atezolizumab is administered intravenously. In
some embodiments, bevacizumab is administered intravenously. In
some embodiments, MOXR0916, atezolizumab, and bevacizumab are
administered intravenously.
[0503] In some embodiments, provided herein is a method of treating
or delaying progression of cancer in an individual comprising
administering to the individual (i) MOXR0916 at a dose of 160 mg,
(ii) atezolizumab at a dose of 1200 mg, and (iii) bevacizumab at a
dose of 15 mg/kg, wherein the cancer is selected from the group
consisting of melanoma, triple-negative breast cancer, ovarian
cancer, renal cell cancer, bladder cancer, non-small cell lung
cancer, gastric cancer, and colorectal cancer. In certain
embodiments, the cancer is renal cell cancer. In certain
embodiments, the cancer is colorectal cancer. In some embodiments,
the MOXR0916, atezolizumab, and bevacizumab are administered on the
same day. In some embodiments, the method further comprises
repeating the administration of MOXR0916 at a dose of 160 mg per
administration, atezolizumab at a dose of 1200 mg per
administration, and bevacizumab at a dose of 15 mg/kg per
administration, and wherein the administration is repeated at an
interval of about 3 weeks or about 21 days between administrations.
In some embodiments, the repeated administrations of the MOXR0916,
the atezolizumab, and the bevacizumab are administered on the same
day. In some embodiments, MOXR0916 is administered intravenously.
In some embodiments, atezolizumab is administered intravenously. In
some embodiments, bevacizumab is administered intravenously. In
some embodiments, MOXR0916, atezolizumab, and bevacizumab are
administered intravenously.
[0504] In some embodiments, provided herein is a method of treating
or delaying progression of cancer in an individual comprising
administering to the individual (i) MOXR0916 at a dose of 320 mg,
(ii) atezolizumab at a dose of 1200 mg, and (iii) bevacizumab at a
dose of 15 mg/kg, wherein the cancer is selected from the group
consisting of melanoma, triple-negative breast cancer, ovarian
cancer, renal cell cancer, bladder cancer, non-small cell lung
cancer, gastric cancer, and colorectal cancer. In certain
embodiments, the cancer is renal cell cancer. In certain
embodiments, the cancer is colorectal cancer. In some embodiments,
the MOXR0916, atezolizumab, and bevacizumab are administered on the
same day. In some embodiments, the method further comprises
repeating the administration of MOXR0916 at a dose of 320 mg per
administration, atezolizumab at a dose of 1200 mg per
administration, and bevacizumab at a dose of 15 mg/kg per
administration, and wherein the administration is repeated at an
interval of about 3 weeks or about 21 days between administrations.
In some embodiments, the repeated administrations of the MOXR0916,
the atezolizumab, and the bevacizumab are administered on the same
day. In some embodiments, MOXR0916 is administered intravenously.
In some embodiments, atezolizumab is administered intravenously. In
some embodiments, bevacizumab is administered intravenously. In
some embodiments, MOXR0916, atezolizumab, and bevacizumab are
administered intravenously.
[0505] In some embodiments, provided herein is a method of treating
or delaying progression of cancer in an individual comprising
administering to the individual (i) MOXR0916 at a dose of 400 mg,
(ii) atezolizumab at a dose of 1200 mg, and (iii) bevacizumab at a
dose of 15 mg/kg, wherein the cancer is selected from the group
consisting of melanoma, triple-negative breast cancer, ovarian
cancer, renal cell cancer, bladder cancer, non-small cell lung
cancer, gastric cancer, and colorectal cancer. In certain
embodiments, the cancer is renal cell cancer. In certain
embodiments, the cancer is colorectal cancer. In some embodiments,
the MOXR0916, atezolizumab, and bevacizumab are administered on the
same day. In some embodiments, the method further comprises
repeating the administration of MOXR0916 at a dose of 400 mg per
administration, atezolizumab at a dose of 1200 mg per
administration, and bevacizumab at a dose of 15 mg/kg per
administration, and wherein the administration is repeated at an
interval of about 3 weeks or about 21 days between administrations.
In some embodiments, the repeated administrations of the MOXR0916,
the atezolizumab, and the bevacizumab are administered on the same
day. In some embodiments, MOXR0916 is administered intravenously.
In some embodiments, atezolizumab is administered intravenously. In
some embodiments, bevacizumab is administered intravenously. In
some embodiments, MOXR0916, atezolizumab, and bevacizumab are
administered intravenously.
[0506] In a further aspect, the invention provides pharmaceutical
formulations comprising any of the anti-OX40 antibodies, anti-VEGF
antibodies, and/or anti-PDL1 antibodies provided herein, e.g., for
use in any of the above therapeutic methods. In one embodiment, a
pharmaceutical formulation comprises any of the anti-OX40
antibodies provided herein and a pharmaceutically acceptable
carrier, and/or (or for use in conjunction with) any of the
anti-PDL1 antibodies provided herein and a pharmaceutically
acceptable carrier, and/or (or for use in conjunction with) any of
the anti-VEGF antibodies provided herein and a pharmaceutically
acceptable carrier. In another embodiment, a pharmaceutical
formulation comprises any of the anti-OX40 antibodies provided
herein and at least one additional therapeutic agent, e.g., as
described below.
[0507] In some embodiments of any of the methods of the invention,
the anti-human OX40 agonist antibodies inhibits tumor immunity by
inhibiting Treg function (e.g., inhibiting the suppressive function
of Tregs), killing OX40 expressing cells (e.g., cells that express
high levels of OX40), increasing effector T cell function and/or
increasing memory T cell function. In some embodiments of any of
the methods of the invention, the anti-human OX40 agonist
antibodies treat cancer by inhibiting Treg function (e.g.,
inhibiting the suppressive function of Tregs), killing OX40
expressing cells (e.g., cells that express high levels of OX40),
increasing effector T cell function and/or increasing memory T cell
function. In some embodiments of any of the methods of the
invention, the anti-human OX40 agonist antibodies enhance immune
function by inhibiting Treg function (e.g., inhibiting the
suppressive function of Tregs), killing OX40 expressing cells
(e.g., cells that express high levels of OX40), increasing effector
T cell function and/or increasing memory T cell function. In some
embodiments of any of the methods of the invention, the anti-human
OX40 agonist antibodies enhance T cell function by inhibiting Treg
function (e.g., inhibiting the suppressive function of Tregs),
killing OX40 expressing cells (e.g., cells that express high levels
of OX40), increasing effector T cell function and/or increasing
memory T cell function.
[0508] In some embodiments of any of the methods, the anti-human
OX40 agonist antibody is a depleting anti-human agonist antibody.
In some embodiments, treatment with the anti-human OX40 agonist
antibody results in cell depletion (e.g., depletion of
OX40-expressing cells, e.g., depletion of cells that express high
levels of OX40). In some embodiments, depletion is by ADCC. In some
embodiments, depletion is by phagocytosis.
[0509] In some embodiments of any of the methods, the anti-human
OX40 agonist antibody inhibits Treg function, e.g., by inhibiting
Treg suppression of effector and/or memory T cell function (in some
embodiments, effector T cell and/or memory T cell proliferation
and/or cytokine secretion), relative to Treg function prior to
administration of the OX40 agonist antibody. In some embodiments of
any of the methods, the anti-human OX40 agonist antibody increases
effector T cell proliferation, relative to effector T cell
proliferation prior to administration of the OX40 agonist antibody.
In some embodiments of any of the methods, the anti-human OX40
agonist antibody increases memory T cell proliferation, relative to
memory T cell proliferation prior to administration of the OX40
agonist antibody. In some embodiments of any of the methods, the
anti-human OX40 agonist antibody increases effector T cell cytokine
production (e.g., gamma interferon production), relative to
effector T cell cytokine production prior to administration of the
OX40 agonist antibody. In some embodiments of any of the methods,
the anti-human OX40 agonist antibody increases memory T cell
cytokine production (e.g., gamma interferon production), relative
to memory T cell cytokine production prior to administration of the
OX40 agonist antibody. In some embodiments of any of the methods,
the anti-human OX40 agonist antibody increases CD4+ effector T cell
proliferation and/or CD8+ effector T cell proliferation relative to
CD4+ effector T cell proliferation and/or CD8+ effector T cell
proliferation prior to administration of the OX40 agonist antibody.
In some embodiments of any of the methods, the anti-human OX40
agonist antibody increases memory T cell proliferation (e.g., CD4+
memory T cell proliferation), relative to memory T cell
proliferation prior to administration of the OX40 agonist antibody.
In some embodiments, the CD4+ effector T cells in the individual
have enhanced proliferation, cytokine secretion and/or cytolytic
activity relative to proliferation, cytokine secretion and/or
cytolytic activity prior to the administration of the anti-human
OX40 agonist antibody.
[0510] In some embodiments of any of the methods of the invention,
the number of CD4+ effector T cells is elevated relative to prior
to administration of the anti-human OX40 agonist antibody. In some
embodiments, CD4+ effector T cell cytokine secretion is elevated
relative to prior to administration of the anti-human OX40 agonist
antibody. In some embodiments of any of the methods, the CD8+
effector T cells in the individual have enhanced proliferation,
cytokine secretion and/or cytolytic activity relative to prior to
the administration of the anti-human OX40 agonist antibody. In some
embodiments, the number of CD8+ effector T cells is elevated
relative to prior to administration of the anti-human OX40 agonist
antibody. In some embodiments, CD8+ effector T cell cytokine
secretion is elevated relative to prior to administration of the
anti-human OX40 agonist antibody.
[0511] In some embodiments of any of the methods of the invention,
the anti-human OX40 agonist antibody binds human effector cells,
e.g., binds Fc.gamma.R expressed by human effector cells. In some
embodiments, the human effector cell performs ADCC effector
function. In some embodiments, the human effector cell performs
phagocytosis effector function.
[0512] In some embodiments of any of the methods of the invention,
the anti-human OX40 agonist antibody comprising a variant IgG1 Fc
polypeptide comprising a mutation that eliminates binding to human
effector cells (e.g., a DANA or N297G mutation) has diminished
activity (e.g., CD4+ effector T cell function, e.g.,
proliferation), relative to anti-human OX40 agonist antibody
comprising native sequence IgG1 Fc portion. In some embodiment, the
anti-human OX40 agonist antibody comprising a variant IgG1 Fc
polypeptide comprising a mutation that eliminates binding to human
effector cells (e.g., a DANA or N297G mutation) does not possess
substantial activity (e.g., CD4+ effector T cell function, e.g.,
proliferation).
[0513] In some embodiments of any of the methods of the invention,
antibody cross-linking is required for anti-human OX40 agonist
antibody function. In some embodiments, function is stimulation of
CD4+ effector T cell proliferation. In some embodiments, antibody
cross-linking is determined by providing anti-human OX40 agonist
antibody adhered on a solid surface (e.g., a cell culture plate).
In some embodiments, antibody cross-linking is determined by
introducing a mutation in the antibody's IgG1 Fc portion (e.g., a
DANA or N297S mutation) and testing function of the mutant
antibody.
[0514] In some embodiments of any of the methods, the memory T
cells in the individual have enhanced proliferation and/or cytokine
secretion relative to prior to the administration of the anti-human
OX40 agonist antibody. In some embodiments, the number of memory T
cells is elevated relative to prior to administration of the
anti-human OX40 agonist antibody. In some embodiments, memory T
cell cytokine secretion (level) is elevated relative to prior to
administration of the anti-human OX40 agonist antibody. In some
embodiments of any of the methods, the Treg in the individual have
decreased inhibition of effector T cell function (e.g.,
proliferation and/or cytokine secretion) relative to prior to the
administration of the anti-human OX40 agonist antibody. In some
embodiments, the number of effector T cells is elevated relative to
prior to administration of the anti-human OX40 agonist antibody. In
some embodiments, effector T cell cytokine secretion (level) is
elevated relative to prior to administration of the anti-human OX40
agonist antibody.
[0515] In some embodiments of any of the methods of the invention,
the number of intratumoral (infiltrating) CD4+ effector T cells
(e.g., total number of CD4+ effector T cells, or e.g., percentage
of CD4+ cells in CD45+ cells) is elevated relative to prior to
administration of the anti-human OX40 agonist antibody. In some
embodiments of any of the methods of the invention, number of
intratumoral (infiltrating) CD4+ effector T cells that express
gamma interferon (e.g., total gamma interferon expressing CD4+
cells, or e.g., percentage of gamma interferon expressing CD4+
cells in total CD4+ cells) is elevated relative to prior to
administration anti-human OX40 agonist antibody.
[0516] In some embodiments of any of the methods of the invention,
the number of intratumoral (infiltrating) CD8+ effector T cells
(e.g., total number of CD8+ effector T cells, or e.g., percentage
of CD8+ in CD45+ cells) is elevated relative to prior to
administration of anti-human OX40 agonist antibody. In some
embodiments of any of the methods of the invention, number of
intratumoral (infiltrating) CD8+ effector T cells that express
gamma interferon (e.g., percentage of CD8+ cells that express gamma
interferon in total CD8+ cells) is increased relative to prior to
administration of anti-human OX40 agonist antibody.
[0517] In some embodiments of any of the methods of the invention,
the number of intratumoral (infiltrating) Treg (e.g., total number
of Treg or e.g., percentage of Fox3p+ cells in CD4+ cells) is
reduced relative to prior to administration of anti-human OX40
agonist antibody.
[0518] In some embodiments of any of the methods of the invention,
administration of anti-human OX40 agonist antibody is in
combination with administration of a tumor antigen. In some
embodiments, the tumor antigen comprises protein. In some
embodiments, the tumor antigen comprises nucleic acid. In some
embodiments, the tumor antigen is a tumor cell.
[0519] In some embodiments of any of the methods of the invention,
a tumor response to treatment may be evaluated. In some
embodiments, RECIST criteria, such as RECIST v1.1, may be used to
evaluate tumor response. These criteria are known in the art and
may be used to measure a patient's response to a treatment; see,
e.g., Eisenhauer, E. A. et al. (2009) Eur. J. Cancer 45:228-247. In
some embodiments, RECIST response criteria may include:
(a) Complete response (CR): disappearance of all target lesions.
Any pathological lymph nodes (whether target or non-target) must
have reduction in short axis to <10 mm; (b) Partial response
(PR): at least a 30% decrease in the sum of diameters of target
lesions, taking as reference the baseline sum of diameters; (c)
Progressive disease (PD): at least a 20% increase in the sum of
diameters of target lesions, taking as reference the smallest sum
on study (nadir), including baseline. In addition to the relative
increase of 20%, the sum must also demonstrate an absolute increase
of at least 5 mm. The appearance of one or more new lesions is also
considered progression; and (d) Stable disease (SD): neither
sufficient shrinkage to qualify for PR nor sufficient increase to
qualify for PD, taking as reference the smallest sum on study.
[0520] In other embodiments, modified RECIST criteria may be used
to evaluate tumor response. Modified Response Evaluation Criteria
in Solid Tumors (RECIST) is derived from RECIST, Version 1.1 (v1.1)
conventions (see, e.g., Eisenhauer, E. A. et al. (2009) Eur. J.
Cancer 45:228-247) and immune-related response criteria (irRC; see,
e.g., Wolchok et al. (2009) Clin. Can. Res. 15:7412-7420; Nishino
et al. (2014) J. Immunother. Can. 2:17; and Nishino et al. (2013)
Clin. Can. Res. 19:3936-3943). Without wishing to be bound to
theory, it is thought that conventional response criteria may not
be adequate to characterize the anti-tumor activity of
immunotherapeutic agents like anti-human OX40 agonist antibodies
and/or anti-PDL1 antibodies, which can produce delayed responses
that may be preceded by initial apparent radiographic progression,
including the appearance of new lesions. Therefore, modified
response criteria have been developed that account for the possible
appearance of new lesions and allow radiological progression to be
confirmed at a subsequent assessment. A summary of the changes
between modified RECIST and RECIST v1.1 is provided in Table B
below.
TABLE-US-00018 TABLE B RECIST v1.1 Modified RECIST New lesions
Define progression New measurable lesions are after baseline added
into the total tumor burden and followed. Non-target May contribute
to the Contribute only in the lesions designation of overall
assessment of a complete progression. response. Radiographic First
instance of .gtoreq.20% Determined only on the basis of progression
increase in the sum measurable disease; may be of diameters or
confirmed by a consecutive unequivocal progression assessment
.gtoreq.4 weeks from the in non-target disease. date first
documented.
[0521] In some embodiments, modified RECIST response criteria may
include:
(a) Complete response (CR): disappearance of all target and
non-target lesions. Lymph nodes that shrink to <10 mm short axis
are considered normal; (b) Partial response (PR): at least a 30%
decrease in the sum of the diameters of all target and all new
measurable lesions, taking as reference the baseline sum of
diameters, in the absence of CR. Note: the appearance of new
measurable lesions is factored into the overall tumor burden, but
does not automatically qualify as progressive disease until the sum
of the diameters increases by .gtoreq.20% when compared with the
sum of the diameters at nadir; (c) Progressive disease (PD): at
least a 20% increase in the sum of diameters of all target and
selected new measurable lesions, taking as reference the smallest
sum on study (nadir SLD; this includes the baseline sum if that is
the smallest on study). In addition to the relative increase of
20%, the sum must also demonstrate an absolute increase of at least
5 mm; and (d) Stable disease (SD): neither sufficient shrinkage to
qualify for PR nor sufficient increase to qualify for PD, taking as
reference the smallest sum of the diameters while on study.
[0522] The assessment of non-target lesions may be captured on the
CRF at each timepoint using standard RECIST v1.1 definitions of CR,
non-CR/non-PD, and PD (unequivocal progression). However, in
determining the overall modified RECIST tumor response, non-target
lesions contribute only to the assessment of a complete response.
Non-target lesions are not considered in the overall definition of
PR, SC, or PD per modified RECIST.
[0523] In some embodiments, new lesions alone do not qualify as
progressive disease. However, their contribution to total tumor
burden may be included in the sum of the diameters, which may be
used to determine the overall modified RECIST tumor response.
[0524] In some embodiments, responsiveness to treatment may refer
to any one or more of: extending survival (including overall
survival and progression free survival); resulting in an objective
response (including a complete response or a partial response); or
improving signs or symptoms of cancer. In some embodiments,
responsiveness may refer to improvement of one or more factors
according to the published set of RECIST guidelines for determining
the status of a tumor in a cancer patient, i.e., responding,
stabilizing, or progressing. For a more detailed discussion of
these guidelines, see Eisenhauer et al., Eur J Cancer 2009; 45:
228-47; Topalian et al., N Engl J Med 2012; 366:2443-54; Wolchok et
al., Clin Can Res 2009; 15:7412-20; and Therasse, P., et al. J.
Natl. Cancer Inst. 92:205-16 (2000). A responsive subject may refer
to a subject whose cancer(s) show improvement, e.g., according to
one or more factors based on RECIST criteria. A non-responsive
subject may refer to a subject whose cancer(s) do not show
improvement, e.g., according to one or more factors based on RECIST
criteria.
[0525] Conventional response criteria may not be adequate to
characterize the anti-tumor activity of immunotherapeutic agents,
which can produce delayed responses that may be preceded by initial
apparent radiological progression, including the appearance of new
lesions. Therefore, modified response criteria have been developed
that account for the possible appearance of new lesions and allow
radiological progression to be confirmed at a subsequent
assessment. Accordingly, in some embodiments, responsiveness may
refer to improvement of one of more factors according to
immune-related response criteria2 (irRC). See, e.g., Wolchok et
al., Clin Can Res 2009; 15:7412-20. In some embodiments, new
lesions are added into the defined tumor burden and followed, e.g.,
for radiological progression at a subsequent assessment. In some
embodiments, presence of non-target lesions are included in
assessment of complete response and not included in assessment of
radiological progression. In some embodiments, radiological
progression may be determined only on the basis of measurable
disease and/or may be confirmed by a consecutive assessment
.gtoreq.4 weeks from the date first documented.
[0526] In some embodiments, responsiveness may include immune
activation. In some embodiments, responsiveness may include
treatment efficacy. In some embodiments, responsiveness may include
immune activation and treatment efficacy.
[0527] In some embodiments of any of the methods of the invention,
the cancer displays human effector cells (e.g., is infiltrated by
human effector cells). Methods for detecting human effector cells
are well known in the art, including, e.g., by IHC. In some
embodiments, the cancer display high levels of human effector
cells. In some embodiments, human effector cells are one or more of
NK cells, macrophages, monocytes. In some embodiments, the cancer
is any cancer described herein. In some embodiments, the cancer is
non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma,
melanoma, breast carcinoma (e.g. triple-negative breast cancer),
gastric cancer, colorectal cancer (CRC), or hepatocellular
carcinoma.
[0528] In some embodiments of any of the methods of the invention,
the cancer displays cells expressing FcR (e.g., is infiltrated by
cells expressing FcR). Methods for detecting FcR are well known in
the art, including, e.g., by IHC. In some embodiments, the cancer
display high levels of cells expressing FcR. In some embodiments,
FcR is Fc.gamma.R. In some embodiments, FcR is activating
Fc.gamma.R. In some embodiments, the cancer is non-small cell lung
cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast
carcinoma (e.g. triple-negative breast cancer), gastric cancer,
colorectal cancer (CRC), or hepatocellular carcinoma.
[0529] In some embodiments, any of the methods of the invention may
further comprise monitoring the responsiveness of the individual to
treatment, e.g., with an anti-human OX40 agonist antibody as
described herein. In some embodiments, monitoring the
responsiveness of an individual to treatment may include measuring
the expression level of one or more marker genes in a sample (e.g.,
a tumor sample) obtained from the individual after treatment. In
some embodiments, the individual may be classified as responsive or
non-responsive to treatment based on the expression level of one or
more marker genes in a sample (e.g., a tumor sample) obtained from
the individual, e.g., as compared with a reference. In some
embodiments, the one or more marker genes may be selected from
CCR5, CD274 (also known as PD-L1), IL-7, TNFRSF14, TGFB1, CD40,
CD4, PRF1, TNFSF4, CD86, CXCL9, CD3E, LAG3, PDCD1, CCL28, GZMB,
IFNg, and IL-2RA, and an increased expression level (e.g., as
compared with a reference) may indicate responsiveness to
treatment. In certain embodiments, increased expression of PD-L1
(e.g., as compared with a reference) may indicate responsiveness to
treatment. In some embodiments, the one or more marker genes may be
selected from CD8b, EOMES, GZMA, GZMB, IFNg, and PRF1, and an
increased expression level (e.g., as compared with a reference) may
indicate responsiveness to treatment. Without wishing to be bound
to theory, it is thought that increased expression of CCR5, CD274
(also known as PD-L1), IL-7, TNFRSF14, TGFB1, CD40, CD4, PRF1,
TNFSF4, CD86, CXCL9, CD3E, LAG3, PDCD1, CCL28, GZMB, IFNg, IL-2RA,
GZMA, CD8b, and/or EOMES may be associated with increased Teff
activity. In other embodiments, the one or more marker genes may be
selected from CCL22, IL-2, RORC, IL-8, CTLA4, and FOXP3, and a
decreased expression level (e.g., as compared with a reference) may
indicate responsiveness to treatment. Without wishing to be bound
to theory, it is thought that decreased expression of CCL22, IL-2,
RORC, IL-8, CTLA4, and/or FOXP3 may be associated with decreased
Treg activity.
[0530] In some embodiments, any of the methods of the invention may
further comprise monitoring efficacy of treatment (e.g., treatment
with an anti-human OX40 agonist antibody as described herein). In
some embodiments, monitoring the efficacy of treatment in an
individual may include measuring the expression level of one or
more marker genes in a sample (e.g., a tumor sample) obtained from
the individual after treatment. In some embodiments, the treatment
may be classified as efficacious based on the expression level of
one or more marker genes in a sample (e.g., a tumor sample)
obtained from the individual, e.g., as compared with a reference.
In some embodiments, the one or more marker genes may be selected
from CCR5, CD274 (also known as PD-L1), IL-7, TNFRSF14, TGFB1,
CD40, CD4, PRF1, TNFSF4, CD86, CXCL9, CD3E, LAG3, PDCD1, CCL28,
GZMB, IFNg, and IL-2RA, and an increased expression level (e.g., as
compared with a reference) may indicate treatment efficacy. In
certain embodiments, increased expression of PD-L1 (e.g., as
compared with a reference) may indicate treatment efficacy. In some
embodiments, the one or more marker genes may be selected from
CD8b, EOMES, GZMA, GZMB, IFNg, and PRF1, and an increased
expression level (e.g., as compared with a reference) may indicate
treatment efficacy. Without wishing to be bound to theory, it is
thought that increased expression of CCR5, CD274 (also known as
PD-L1), IL-7, TNFRSF14, TGFB1, CD40, CD4, PRF1, TNFSF4, CD86,
CXCL9, CD3E, LAG3, PDCD1, CCL28, GZMB, IFNg, IL-2RA, GZMA, CD8b,
and/or EOMES may be associated with increased Teff activity. In
other embodiments, the one or more marker genes may be selected
from CCL22, IL-2, RORC, IL-8, CTLA4, and FOXP3, and a decreased
expression level (e.g., as compared with a reference) may indicate
treatment efficacy. Without wishing to be bound to theory, it is
thought that decreased expression of CCL22, IL-2, RORC, IL-8,
CTLA4, and/or FOXP3 may be associated with decreased Treg
activity.
[0531] In some embodiments, the expression level of one or more
marker genes described herein is compared to a reference. In some
embodiments, a reference may include a biopsy obtained from the
individual before treatment, a biopsy obtained from an untreated
individual, or a reference or baseline value. In some embodiments,
the reference is the average, mean, or median level of expression
of the corresponding marker gene(s) in samples obtained from
individuals that have cancer (e.g., the same type of cancer as the
individual receiving treatment). In some embodiments, the reference
is the average, mean, or median level of expression of the
corresponding marker gene in samples from other subjects having
cancer who are not responsive to the OX40 agonist treatment after
receiving treatment. For example, a set of samples obtained from
cancers having a shared characteristic (e.g., the same cancer type
and/or stage, or exposure to a common treatment such as an OX40
agonist) may be studied from a population, such as with a clinical
outcome study. This set may be used to derive a reference, e.g., a
reference number, to which a subject's sample may be compared.
[0532] In some embodiments, expression level of an mRNA or protein
may be normalized to the expression level of a reference gene.
Normalizing the expression level of a particular gene to a
reference is thought to enhance reproducibility across samples by
factoring differences in sample size and/or mRNA/protein
extraction. In these examples, expression level relative to the
reference is measured. In some embodiments, multiple reference
genes may be used, either singly or in aggregate (e.g., by
averaging). In other embodiments, expression level of an mRNA or
protein may refer to absolute expression level.
[0533] In some embodiments, a reference gene may be a housekeeping
gene. A housekeeping gene is thought to be constitutively expressed
in a cell in normal and/or pathological states, such as a gene
encoding a protein required for basic cellular function and/or
maintenance. Housekeeping genes are typically used as a reference
to ensure they will be expressed at a detectable and/or
reproducible level across multiple samples. Exemplary housekeeping
genes and further description of the use of such genes as a
reference may be found, for example, in de Kok, J. B., et al.
(2005) Lab Invest. 85(1):154-9.
[0534] Certain aspects of the present disclosure relate to
measurement of the expression level of one or more genes in a
sample. In some embodiments, a sample may include leukocytes. In
some embodiments, the sample may be a tumor sample. A tumor sample
may include cancer cells, lymphocytes, leukocytes, stroma, blood
vessels, connective tissue, basal lamina, and any other cell type
in association with the tumor. In some embodiments, the sample is a
tumor tissue sample containing tumor-infiltrating leukocytes. As
used herein, any leukocyte associated with a tumor may be
considered a tumor-infiltrating leukocyte. Examples of
tumor-infiltrating leukocytes include without limitation T
lymphocytes (such as CD8+ T lymphocytes and/or CD4+ T lymphocytes),
B lymphocytes, or other bone marrow-lineage cells including
granulocytes (neutrophils, eosinophils, basophils), monocytes,
macrophages, dendritic cells (i.e., interdigitating dendritic
cells), histiocytes, and natural killer cells. In some embodiments,
a tumor-infiltrating leukocyte may be associated with cancer cells
of a tumor. In some embodiments, a tumor-infiltrating leukocyte may
be associated with tumor stroma. In some embodiments, the tumor
samples are enriched for tumor area by macrodissection.
[0535] In some embodiments, the sample may be processed to separate
or isolate one or more cell types (e.g., leukocytes). In some
embodiments, the sample may be used without separating or isolating
cell types. A tumor sample may be obtained from a subject by any
method known in the art, including without limitation a biopsy,
endoscopy, or surgical procedure. In some embodiments, a tumor
sample may be prepared by methods such as freezing, fixation (e.g.,
by using formalin or a similar fixative), and/or embedding in
paraffin wax. In some embodiments, a tumor sample may be sectioned.
In some embodiments, a fresh tumor sample (i.e., one that has not
been prepared by the methods described above) may be used. In some
embodiments, a sample may be prepared by incubation in a solution
to preserve mRNA and/or protein integrity. A tumor sample
containing leukocytes may be assayed by any technique described
herein for measuring marker gene expression level.
[0536] Certain aspects of the present disclosure relate to
measuring the expression level of one or more marker genes. Any
suitable method for measuring gene expression known in the art may
be used. In some embodiments, expression level may refer to mRNA
expression level. mRNA expression level may be measured by many
methods. Such methods may quantify the copies of a specific mRNA
present in a sample by measuring the amount of hybridization to an
mRNA-specific probe. Other methods may amplify mRNA, or cDNA
generated from mRNA, and quantify the amount of amplicon generated
to extrapolate how much mRNA was present in a sample. Yet other
methods may involve next-generation sequencing of part or all of
mRNA transcripts, or cDNA generated from mRNA, then quantifying the
number of sequences detected that correspond to particular gene(s).
In some embodiments, mRNA expression level is measured by
quantitative PCR, semi-quantitative PCR, nucleotide microarray,
RNA-seq, in situ hybridization, and/or Northern blotting.
[0537] In some embodiments, expression level may refer to protein
expression level. Protein expression level may be measured by many
methods. Such methods may quantify proteins present in a sample by
using a probe that specifically binds to a particular protein, such
as an antibody, then detecting the amount of specific binding in a
sample. Other methods may fragment proteins into short peptides,
then detect these peptides and quantify how many peptides
correspond to particular protein(s). In some embodiments, protein
expression level is measured by Western blotting, peptide
microarray, immunohistochemistry, flow cytometry, and/or mass
spectrometry.
[0538] An "individual" according to any of the above embodiments is
preferably a human.
[0539] Antibodies of the invention can be used either alone or in
combination with other agents in a therapy. For instance, an
antibody of the invention may be co-administered with at least one
additional therapeutic agent.
[0540] Such combination therapies noted above encompass combined
administration (where two or more therapeutic agents are included
in the same or separate formulations), and separate administration,
in which case, administration of the antibody of the invention can
occur prior to, simultaneously, and/or following, administration of
the additional therapeutic agent or agents. In one embodiment,
administration of the anti-OX40 antibody and administration of an
additional therapeutic agent occur within about one month, or
within about one, two or three weeks, or within about one, two,
three, four, five, or six days, of each other. Antibodies of the
invention can also be used in combination with radiation
therapy.
[0541] In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with a chemotherapy or chemotherapeutic
agent. In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with a radiation therapy or
radiotherapeutic agent. In some embodiments, an anti-human OX40
agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with a targeted
therapy or targeted therapeutic agent. In some embodiments, an
anti-human OX40 agonist antibody and/or anti-PDL1 antibody of the
present disclosure may be administered in conjunction with an
immunotherapy or immunotherapeutic agent, for example a monoclonal
antibody.
[0542] In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with a PARP inhibitor (e.g.,
Olaparanib, Rucaparib, Niraparib, Cediranib, BMN673, Veliparib),
Trabectedin, nab-paclitaxel (albumen-bound paclitaxel, ABRAXANE),
Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus,
fluoropyrimidine (e.g., FOLFOX, FOLFIRI), IFL, regorafenib,
Reolysin, Alimta, Zykadia, Sutent, Torisel (temsirolimus), Inlyta
(axitinib, Pfizer), Afinitor (everolimus, Novartis), Nexavar
(sorafenib, Onyx/Bayer), Votrient, Pazopanib, axitinib, IMA-901,
AGS-003, cabozantinib, Vinflunine, Hsp90 inhibitor (e.g.,
apatorsin), Ad-GM-CSF (CT-0070), Temazolomide, IL-2, IFNa,
vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide,
azacytidine, lenalidomide, bortezomid (VELCADE), amrubicine,
carfilzomib, pralatrexate, and/or enzastaurin.
[0543] In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with an agonist directed against an
activating co-stimulatory molecule. In some embodiments, an
activating co-stimulatory molecule may include CD40, CD226, CD28,
GITR, CD137, CD27, HVEM, or CD127. In some embodiments, the agonist
directed against an activating co-stimulatory molecule is an
agonist antibody that binds to CD40, CD226, CD28, OX40, GITR,
CD137, CD27, HVEM, or CD127. In some embodiments, an anti-human
OX40 agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with an antagonist
directed against an inhibitory co-stimulatory molecule. In some
embodiments, an inhibitory co-stimulatory molecule may include
CTLA-4 (also known as CD152), PD-1, TIM-3, BTLA, VISTA, LAG-3,
B7-H3, B7-H4, IDO, TIGIT, MICA/B, or arginase. In some embodiments,
the antagonist directed against an inhibitory co-stimulatory
molecule is an antagonist antibody that binds to CTLA-4, PD-1,
TIM-3, BTLA, VISTA, LAG-3 (e.g., LAG-3-IgG fusion protein
(IMP321)), B7-H3, B7-H4, IDO, TIGIT, MICA/B, or arginase.
[0544] In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with an antagonist directed against
CTLA-4 (also known as CD152), e.g., a blocking antibody. In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with ipilimumab (also known as MDX-010, MDX-101, or
Yervoy.RTM.). In some embodiments, an anti-human OX40 agonist
antibody and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with tremelimumab (also known as
ticilimumab or CP-675,206). In some embodiments, an anti-human OX40
agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with an antagonist
directed against B7-H3 (also known as CD276), e.g., a blocking
antibody. In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with MGA271. In some embodiments, an
anti-human OX40 agonist antibody and/or anti-PDL1 antibody of the
present disclosure may be administered in conjunction with an
antagonist directed against a TGF beta, e.g., metelimumab (also
known as CAT-192), fresolimumab (also known as GC1008), or
LY2157299.
[0545] In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with a treatment comprising adoptive
transfer of a T cell (e.g., a cytotoxic T cell or CTL) expressing a
chimeric antigen receptor (CAR). In some embodiments, an anti-human
OX40 agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with UCART19. In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with WT128z. In some embodiments, an anti-human OX40
agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with KTE-C19 (Kite).
In some embodiments, an anti-human OX40 agonist antibody and/or
anti-PDL1 antibody of the present disclosure may be administered in
conjunction with CTL019 (Novartis). In some embodiments, an
anti-human OX40 agonist antibody and/or anti-PDL1 antibody of the
present disclosure may be administered in conjunction with a
treatment comprising adoptive transfer of a T cell comprising a
dominant-negative TGF beta receptor, e.g, a dominant-negative TGF
beta type II receptor. In some embodiments, an anti-human OX40
agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with a treatment
comprising a HERCREEM protocol (see, e.g., ClinicalTrials.gov
Identifier NCT00889954).
[0546] In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with an antagonist directed against
CD19. In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with MOR00208. In some embodiments, an
anti-human OX40 agonist antibody and/or anti-PDL1 antibody of the
present disclosure may be administered in conjunction with an
antagonist directed against CD38. In some embodiments, an
anti-human OX40 agonist antibody and/or anti-PDL1 antibody of the
present disclosure may be administered in conjunction with
daratumumab.
[0547] In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with an agonist directed against CD137
(also known as TNFRSF9, 4-1BB, or ILA), e.g., an activating
antibody. In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with urelumab (also known as
BMS-663513). In some embodiments, an anti-human OX40 agonist
antibody and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with an agonist directed against CD40,
e.g., an activating antibody. In some embodiments, an anti-human
OX40 agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with CP-870893. In
some embodiments, an anti-human OX40 agonist antibody and/or
anti-PDL1 antibody of the present disclosure may be administered in
conjunction with an agonist directed against OX40 (also known as
CD134), e.g., an activating antibody. In some embodiments, an
anti-human OX40 agonist antibody may be administered in conjunction
with a different anti-OX40 antibody (e.g., AgonOX). In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with an agonist directed against CD27, e.g., an
activating antibody. In some embodiments, an anti-human OX40
agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with CDX-1127. In
some embodiments, an anti-human OX40 agonist antibody and/or
anti-PDL1 antibody of the present disclosure may be administered in
conjunction with an antagonist directed against
indoleamine-2,3-dioxygenase (IDO). In some embodiments, with the
IDO antagonist is 1-methyl-D-tryptophan (also known as 1-D-MT).
[0548] In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with an agonist directed against CD137
(also known as TNFRSF9, 4-1BB, or ILA), e.g., an activating
antibody. In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with urelumab (also known as
BMS-663513). In some embodiments, an anti-human OX40 agonist
antibody and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with an agonist directed against CD40,
e.g., an activating antibody. In some embodiments, an anti-human
OX40 agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with CP-870893 or
R07009789. In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with an agonist directed against OX40
(also known as CD134), e.g., an activating antibody.). In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with an agonist directed against CD27, e.g., an
activating antibody. In some embodiments, an anti-human OX40
agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with CDX-1127 (also
known as varlilumab). In some embodiments, an anti-human OX40
agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with an antagonist
directed against indoleamine-2,3-dioxygenase (IDO). In some
embodiments, with the IDO antagonist is 1-methyl-D-tryptophan (also
known as 1-D-MT). In some embodiments, the IDO antagonist is an IDO
antagonist shown in WO2010/005958 (the contents of which are
expressly incorporated by record herein). In some embodiments the
IDO antagonist is
4-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-(3-bromo-4-fluorophenyl)-N'-hy-
droxy-1,2,5-oxadiazole-3-carboximidamide (e.g., as described in
Example 23 of WO2010/005958). In some embodiments the IDO
antagonist is
##STR00001##
In some embodiments, the IDO antagonist is INCB24360. In some
embodiments, the IDO antagonist is Indoximod (the D isomer of
1-methyl-tryptophan). In some embodiments, an anti-human OX40
agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with an antibody-drug
conjugate. In some embodiments, the antibody-drug conjugate
comprises mertansine or monomethyl auristatin E (MMAE). In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with an anti-NaPi2b antibody-MMAE conjugate (also known
as DNIB0600A, RG7599 or lifastuzumab vedotin). In some embodiments,
an anti-human OX40 agonist antibody and/or anti-PDL1 antibody of
the present disclosure may be administered in conjunction with
trastuzumab emtansine (also known as T-DM1, ado-trastuzumab
emtansine, or KADCYLA.RTM., Genentech). In some embodiments, an
anti-human OX40 agonist antibody and/or anti-PDL1 antibody of the
present disclosure may be administered in conjunction with an
anti-MUC16 antibody-MMAE conjugate, DMUC5754A. In some embodiments,
an anti-human OX40 agonist antibody and/or anti-PDL1 antibody of
the present disclosure may be administered in conjunction with an
anti-MUC16 antibody-MMAE conjugate, DMUC4064A. In some embodiments,
an anti-human OX40 agonist antibody and/or anti-PDL1 antibody of
the present disclosure may be administered in conjunction with an
antibody-drug conjugate targeting the endothelin B receptor
(EDNBR), e.g., an antibody directed against EDNBR conjugated with
MMAE. In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with an antibody-drug conjugate
targeting the lymphocyte antigen 6 complex, locus E (Ly6E), e.g.,
an antibody directed against Ly6E conjugated with MMAE, (also known
as DLYE5953A). In some embodiments, an anti-human OX40 agonist
antibody and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with polatuzumab vedotin. In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with an antibody-drug conjugate targeting CD30. In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with ADCETRIS (also known as brentuximab vedotin). In
some embodiments, an anti-human OX40 agonist antibody and/or
anti-PDL1 antibody of the present disclosure may be administered in
conjunction with polatuzumab vedotin.
[0549] In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with an angiogenesis inhibitor. In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with an antibody directed against a VEGF, e.g., VEGF-A.
In some embodiments, an anti-human OX40 agonist antibody and/or
anti-PDL1 antibody of the present disclosure may be administered in
conjunction with bevacizumab (also known as AVASTIN.RTM.,
Genentech). In some embodiments, an anti-human OX40 agonist
antibody and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with an antibody directed against
angiopoietin 2 (also known as Ang2). In some embodiments, an
anti-human OX40 agonist antibody and/or anti-PDL1 antibody of the
present disclosure may be administered in conjunction with
MEDI3617. In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with an antibody directed against
VEGFR2. In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with ramucirumab. In some embodiments,
an anti-human OX40 agonist antibody and/or anti-PDL1 antibody of
the present disclosure may be administered in conjunction with a
VEGF Receptor fusion protein. In some embodiments, an anti-human
OX40 agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with aflibercept. In
some embodiments, an anti-human OX40 agonist antibody and/or
anti-PDL1 antibody of the present disclosure may be administered in
conjunction with ziv-aflibercept (also known as VEGF Trap or
Zaltrap.RTM.). In some embodiments, an anti-human OX40 agonist
antibody and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with a bispecific antibody directed
against VEGF and Ang2. In some embodiments, an anti-human OX40
agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with RG7221 (also
known as vanucizumab). In some embodiments, an anti-human OX40
agonist antibody may be administered in conjunction with
bevacizumab and a PD-1 axis binding antagonist (e.g., a PD-1
binding antagonist such as an anti-PD-1 antibody, a PD-L1 binding
antagonist such as an anti-PD-L1 antibody, and a PD-L2 binding
antagonist such as an anti-PD-L2 antibody). In some embodiments, an
anti-human OX40 agonist antibody and/or anti-PDL1 antibody of the
present disclosure may be administered in conjunction with
bevacizumab and MDX-1106 (nivolumab, OPDIVO). In some embodiments,
an anti-human OX40 agonist antibody and/or anti-PDL1 antibody of
the present disclosure may be administered in conjunction with
bevacizumab and Merck 3475 (MK-3475, pembrolizumab, KEYTRUDA). In
some embodiments, an anti-human OX40 agonist antibody and/or
anti-PDL1 antibody of the present disclosure may be administered in
conjunction with bevacizumab and CT-011 (Pidilizumab). In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with bevacizumab and YW243.55.S70. In some embodiments,
an anti-human OX40 agonist antibody may be administered in
conjunction with bevacizumab and MPDL3280A. In some embodiments, an
anti-human OX40 agonist antibody and/or anti-PDL1 antibody of the
present disclosure may be administered in conjunction with
bevacizumab and MEDI4736. In some embodiments, an anti-human OX40
agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with bevacizumab and
MDX-1105.
[0550] In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with an antineoplastic agent. In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with an agent targeting CSF-1R (also known as M-CSFR or
CD115). In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with anti-CSF-1R antibody (also known
as IMC-CS4 or LY3022855) In some embodiments, an anti-human OX40
agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with anti-CSF-1R
antibody, RG7155 (also known as R05509554 or emactuzumab). In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with an interferon, for example interferon alpha or
interferon gamma. In some embodiments, an anti-human OX40 agonist
antibody and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with Roferon-A (also known as
recombinant Interferon alpha-2a). In some embodiments, an
anti-human OX40 agonist antibody and/or anti-PDL1 antibody of the
present disclosure may be administered in conjunction with GM-CSF
(also known as recombinant human granulocyte macrophage colony
stimulating factor, rhu GM-CSF, sargramostim, or Leukine.RTM.). In
some embodiments, an anti-human OX40 agonist antibody and/or
anti-PDL1 antibody of the present disclosure may be administered in
conjunction with IL-2 (also known as aldesleukin or
Proleukin.RTM.). In some embodiments, an anti-human OX40 agonist
antibody and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with IL-12. In some embodiments, an
anti-human OX40 agonist antibody and/or anti-PDL1 antibody of the
present disclosure may be administered in conjunction with IL27. In
some embodiments, an anti-human OX40 agonist antibody and/or
anti-PDL1 antibody of the present disclosure may be administered in
conjunction with IL-15. In some embodiments, an anti-human OX40
agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with ALT-803. In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with an antibody targeting CD20. In some embodiments,
the antibody targeting CD20 is obinutuzumab (also known as GA101 or
Gazyva.RTM.) or rituximab. In some embodiments, an anti-human OX40
agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with an antibody
targeting GITR. In some embodiments, the antibody targeting GITR is
TRX518. In some embodiments, the antibody targeting GITR is MK04166
(Merck).
[0551] In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with an inhibitor of Bruton's tyrosine
kinase (BTK). In some embodiments, an anti-human OX40 agonist
antibody and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with ibrutinib. In some embodiments, an
anti-human OX40 agonist antibody and/or anti-PDL1 antibody of the
present disclosure may be administered in conjunction with an
inhibitor of Isocitrate dehydrogenase 1 (IDH1) and/or Isocitrate
dehydrogenase 2 (IDH2). In some embodiments, an anti-human OX40
agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with AG-120
(Agios).
[0552] In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with a cancer vaccine. In some
embodiments, the cancer vaccine is a peptide cancer vaccine, which
in some embodiments is a personalized peptide vaccine. In some
embodiments the peptide cancer vaccine is a multivalent long
peptide, a multi-peptide, a peptide cocktail, a hybrid peptide, or
a peptide-pulsed dendritic cell vaccine (see, e.g., Yamada et al.,
Cancer Sci, 104:14-21, 2013). In some embodiments, an anti-human
OX40 agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with an adjuvant. In
some embodiments, an anti-human OX40 agonist antibody and/or
anti-PDL1 antibody of the present disclosure may be administered in
conjunction with a treatment comprising a TLR agonist, e.g.,
Poly-ICLC (also known as Hiltonol.RTM.), LPS, MPL, or CpG ODN. In
some embodiments, an anti-human OX40 agonist antibody and/or
anti-PDL1 antibody of the present disclosure may be administered in
conjunction with tumor necrosis factor (TNF) alpha. In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with IL-1. In some embodiments, an anti-human OX40
agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with HMGB1. In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with an IL-10 antagonist. In some embodiments, an
anti-human OX40 agonist antibody and/or anti-PDL1 antibody of the
present disclosure may be administered in conjunction with an IL-4
antagonist. In some embodiments, an anti-human OX40 agonist
antibody and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with an IL-13 antagonist. In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with an IL-17 antagonist. In some embodiments, an
anti-human OX40 agonist antibody and/or anti-PDL1 antibody of the
present disclosure may be administered in conjunction with an HVEM
antagonist. In some embodiments, an anti-human OX40 agonist
antibody and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with an ICOS agonist, e.g., by
administration of ICOS-L, or an agonistic antibody directed against
ICOS. In some embodiments, an anti-human OX40 agonist antibody may
be administered in conjunction with a treatment targeting CX3CL1.
In some embodiments, an anti-human OX40 agonist antibody and/or
anti-PDL1 antibody of the present disclosure may be administered in
conjunction with a treatment targeting CXCL9. In some embodiments,
an anti-human OX40 agonist antibody and/or anti-PDL1 antibody of
the present disclosure may be administered in conjunction with a
treatment targeting CXCL10. In some embodiments, an anti-human OX40
agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with a treatment
targeting CCL5. In some embodiments, an anti-human OX40 agonist
antibody and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with an LFA-1 or ICAM1 agonist. In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with a Selectin agonist.
[0553] In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with an inhibitor of B-Raf. In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with vemurafenib (also known as Zelboraf.RTM.). In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with dabrafenib (also known as Tafinlar.RTM.). In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with encorafenib (LGX818).
[0554] In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with an EGFR inhibitor. In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with erlotinib (also known as Tarceva.RTM.). In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with an inhibitor of EGFR-T790M. In some embodiments,
an anti-human OX40 agonist antibody and/or anti-PDL1 antibody of
the present disclosure may be administered in conjunction with
gefitinib. In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with afatinib. In some embodiments, an
anti-human OX40 agonist antibody and/or anti-PDL1 antibody of the
present disclosure may be administered in conjunction with
cetuximab (also known as Erbitux.RTM.). In some embodiments, an
anti-human OX40 agonist antibody and/or anti-PDL1 antibody of the
present disclosure may be administered in conjunction with
panitumumab (also known as Vectibix.RTM.). In some embodiments, an
anti-human OX40 agonist antibody and/or anti-PDL1 antibody of the
present disclosure may be administered in conjunction with
rociletinib. In some embodiments, an anti-human OX40 agonist
antibody and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with AZD9291. In some embodiments, an
anti-human OX40 agonist antibody and/or anti-PDL1 antibody of the
present disclosure may be administered in conjunction with an
inhibitor of a MEK, such as MEK1 (also known as MAP2K1) and/or MEK2
(also known as MAP2K2). In some embodiments, an anti-human OX40
agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with cobimetinib
(also known as GDC-0973 or XL-518). In some embodiments, an
anti-human OX40 agonist antibody and/or anti-PDL1 antibody of the
present disclosure may be administered in conjunction with
trametinib (also known as Mekinist.RTM.). In some embodiments, an
anti-human OX40 agonist antibody and/or anti-PDL1 antibody of the
present disclosure may be administered in conjunction with
binimetinib.
[0555] In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction an inhibitor of B-Raf (e.g.,
vemurafenib or dabrafenib) and an inhibitor of MEK (e.g., MEK1
and/or MEK2 (e.g., cobimetinib or trametinib). In some embodiments,
an anti-human OX40 agonist antibody and/or anti-PDL1 antibody of
the present disclosure may be administered in conjunction with an
inhibitor of ERK (e.g., ERK1/2). In some embodiments, an anti-human
OX40 agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with GDC-0994). In
some embodiments, an anti-human OX40 agonist antibody and/or
anti-PDL1 antibody of the present disclosure may be administered in
conjunction with an inhibitor of B-Raf, an inhibitor of MEK, and an
inhibitor of ERK1/2. In some embodiments, an anti-human OX40
agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with an inhibitor of
EGFR, an inhibitor of MEK, and an inhibitor of ERK1/2. In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with one or more MAP kinase pathway inhibitor. In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with CK127. In some embodiments, an anti-human OX40
agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with an inhibitor of
K-Ras.
[0556] In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with an inhibitor of c-Met. In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with onartuzumab (also known as MetMAb). In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with an inhibitor of anaplatic lymphoma kinase (ALK).
In some embodiments, an anti-human OX40 agonist antibody and/or
anti-PDL1 antibody of the present disclosure may be administered in
conjunction with AF802 (also known as CH5424802 or alectinib). In
some embodiments, an anti-human OX40 agonist antibody and/or
anti-PDL1 antibody of the present disclosure may be administered in
conjunction with crizotinib. In some embodiments, an anti-human
OX40 agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with ceritinib. In
some embodiments, an anti-human OX40 agonist antibody and/or
anti-PDL1 antibody of the present disclosure may be administered in
conjunction with an inhibitor of a phosphatidylinositol 3-kinase
(PI3K). In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with buparlisib (BKM-120). In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with pictilisib (also known as GDC-0941). In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with buparlisib (also known as BKM-120). In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with perifosine (also known as KRX-0401). In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with a delta-selective inhibitor of a
phosphatidylinositol 3-kinase (PI3K). In some embodiments, an
anti-human OX40 agonist antibody and/or anti-PDL1 antibody of the
present disclosure may be administered in conjunction with
idelalisib (also known as GS-1101 or CAL-101). In some embodiments,
an anti-human OX40 agonist antibody and/or anti-PDL1 antibody of
the present disclosure may be administered in conjunction with
taselisib (also known as GDC-0032). In some embodiments, an
anti-human OX40 agonist antibody and/or anti-PDL1 antibody of the
present disclosure may be administered in conjunction with BYL-719.
In some embodiments, an anti-human OX40 agonist antibody and/or
anti-PDL1 antibody of the present disclosure may be administered in
conjunction with an inhibitor of an Akt. In some embodiments, an
anti-human OX40 agonist antibody and/or anti-PDL1 antibody of the
present disclosure may be administered in conjunction with MK2206.
In some embodiments, an anti-human OX40 agonist antibody and/or
anti-PDL1 antibody of the present disclosure may be administered in
conjunction with GSK690693. In some embodiments, an anti-human OX40
agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with ipatasertib
(also known as GDC-0068). In some embodiments, an anti-human OX40
agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with an inhibitor of
mTOR. In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with sirolimus (also known as
rapamycin). In some embodiments, an anti-human OX40 agonist
antibody and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with temsirolimus (also known as
CCI-779 or Torisel.RTM.). In some embodiments, an anti-human OX40
agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with everolimus (also
known as RAD001). In some embodiments, an anti-human OX40 agonist
antibody and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with ridaforolimus (also known as
AP-23573, MK-8669, or deforolimus). In some embodiments, an
anti-human OX40 agonist antibody and/or anti-PDL1 antibody of the
present disclosure may be administered in conjunction with OSI-027.
In some embodiments, an anti-human OX40 agonist antibody and/or
anti-PDL1 antibody of the present disclosure may be administered in
conjunction with AZD8055. In some embodiments, an anti-human OX40
agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with INK128. In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with a dual PI3K/mTOR inhibitor. In some embodiments,
an anti-human OX40 agonist antibody and/or anti-PDL1 antibody of
the present disclosure may be administered in conjunction with
XL765. In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with GDC-0980. In some embodiments, an
anti-human OX40 agonist antibody and/or anti-PDL1 antibody of the
present disclosure may be administered in conjunction with BEZ235
(also known as NVP-BEZ235). In some embodiments, an anti-human OX40
agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with BGT226. In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with GSK2126458. In some embodiments, an anti-human
OX40 agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with PF-04691502. In
some embodiments, an anti-human OX40 agonist antibody and/or
anti-PDL1 antibody of the present disclosure may be administered in
conjunction with PF-05212384 (also known as PKI-587).
[0557] In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with an agent that selectively degrades
the estrogen receptor. In some embodiments, an anti-human OX40
agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with GDC-0927. In
some embodiments, an anti-human OX40 agonist antibody and/or
anti-PDL1 antibody of the present disclosure may be administered in
conjunction with an inhibitor of HER3. In some embodiments, an
anti-human OX40 agonist antibody and/or anti-PDL1 antibody of the
present disclosure may be administered in conjunction with
duligotuzumab. In some embodiments, an anti-human OX40 agonist
antibody and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with an inhibitor of LSD1. In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with an inhibitor of MDM2. In some embodiments, an
anti-human OX40 agonist antibody and/or anti-PDL1 antibody of the
present disclosure may be administered in conjunction with an
inhibitor of BCL2. In some embodiments, an anti-human OX40 agonist
antibody and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with venetoclax. In some embodiments,
an anti-human OX40 agonist antibody and/or anti-PDL1 antibody of
the present disclosure may be administered in conjunction with an
inhibitor of CHK1. In some embodiments, an anti-human OX40 agonist
antibody and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with GDC-0575. In some embodiments, an
anti-human OX40 agonist antibody and/or anti-PDL1 antibody of the
present disclosure may be administered in conjunction with an
inhibitor of activated hedgehog signaling pathway. In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with ERIVEDGE.
[0558] In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with radiation therapy. In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with gemcitabine. In some embodiments, an anti-human
OX40 agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with nab-paclitaxel
(ABRAXANE). In some embodiments, an anti-human OX40 agonist
antibody and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with trastuzumab. In some embodiments,
an anti-human OX40 agonist antibody and/or anti-PDL1 antibody of
the present disclosure may be administered in conjunction with
TVEC. In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with IL27. In some embodiments, an
anti-human OX40 agonist antibody and/or anti-PDL1 antibody of the
present disclosure may be administered in conjunction with
cyclophosphamide. In some embodiments, an anti-human OX40 agonist
antibody and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with an agent that recruits T cells to
the tumor. In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with lirilumab (IPH2102/BMS-986015). In
some embodiments, an anti-human OX40 agonist antibody and/or
anti-PDL1 antibody of the present disclosure may be administered in
conjunction with Idelalisib. In some embodiments, an anti-human
OX40 agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with an antibody that
targets CD3 and CD20. In some embodiments, an anti-human OX40
agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with REGN1979. In
some embodiments, an anti-human OX40 agonist antibody and/or
anti-PDL1 antibody of the present disclosure may be administered in
conjunction with an antibody that targets CD3 and CD19. In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with blinatumomab.
[0559] In some embodiments, an anti-human OX40 agonist antibody
and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with an oncolytic virus. In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with carboplatin and nab-paclitaxel. In some
embodiments, an anti-human OX40 agonist antibody and/or anti-PDL1
antibody of the present disclosure may be administered in
conjunction with carboplatin and paclitaxel. In some embodiments,
an anti-human OX40 agonist antibody and/or anti-PDL1 antibody of
the present disclosure may be administered in conjunction with
cisplatin and pemetrexed. In some embodiments, an anti-human OX40
agonist antibody and/or anti-PDL1 antibody of the present
disclosure may be administered in conjunction with cisplatin and
gemcitabine. In some embodiments, an anti-human OX40 agonist
antibody and/or anti-PDL1 antibody of the present disclosure may be
administered in conjunction with FOLFOX. In some embodiments, an
anti-human OX40 agonist antibody and/or anti-PDL1 antibody of the
present disclosure may be administered in conjunction with
FOLFIRI.
[0560] Such combination therapies noted above encompass combined
administration (where two or more therapeutic agents are included
in the same or separate formulations), and separate administration,
in which case, administration of the one or more antibodies of the
invention can occur prior to, simultaneously, and/or following,
administration of the additional therapeutic agent and/or adjuvant.
Antibodies of the invention can also be used in combination with
radiation therapy.
[0561] An antibody of the invention (and any additional therapeutic
agent) can be administered by any suitable means, including
parenteral, intrapulmonary, and intranasal, and, if desired for
local treatment, intralesional administration. Parenteral infusions
include intramuscular, intravenous, intraarterial, intraperitoneal,
or subcutaneous administration. Dosing can be by any suitable
route, e.g. by injections, such as intravenous or subcutaneous
injections, depending in part on whether the administration is
brief or chronic. Various dosing schedules including but not
limited to single or multiple administrations over various
time-points, bolus administration, and pulse infusion are
contemplated herein.
[0562] In certain embodiments, the antibody is administered
intravenously. In some embodiments, the antibody is administered by
intravenous infusion. For example, the antibody may be delivered
via intravenous infusion over approximately 90 minutes,
approximately 60 minutes, or approximately 30 minutes. In some
embodiments, if a patient tolerates an infusion over a particular
duration (e.g., a 90 minute infusion), subsequent infusions may be
administered over a shorter duration (e.g., 30 or 60 minutes).
Infusions may be slowed or interrupted for infusion-associated
symptoms.
[0563] Antibodies of the invention would be formulated, dosed, and
administered in a fashion consistent with good medical practice.
Factors for consideration in this context include the particular
disorder being treated, the particular mammal being treated, the
clinical condition of the individual patient, the cause of the
disorder, the site of delivery of the agent, the method of
administration, the scheduling of administration, and other factors
known to medical practitioners. The antibody need not be, but is
optionally formulated with one or more agents currently used to
prevent or treat the disorder in question. The effective amount of
such other agents depends on the amount of antibody present in the
formulation, the type of disorder or treatment, and other factors
discussed above. These are generally used in the same dosages and
with administration routes as described herein, or about from 1 to
99% of the dosages described herein, or in any dosage and by any
route that is empirically/clinically determined to be
appropriate.
[0564] For the prevention or treatment of disease, the appropriate
dosage of an antibody of the invention (when used alone or in
combination with one or more other additional therapeutic agents)
will depend on the type of disease to be treated, the type of
antibody, the severity and course of the disease, whether the
antibody is administered for preventive or therapeutic purposes,
previous therapy, the patient's clinical history and response to
the antibody, and the discretion of the attending physician. The
antibody is suitably administered to the patient at one time or
over a series of treatments. Depending on the type and severity of
the disease, about 1 .mu.g/kg to 40 mg/kg of antibody can be an
initial candidate dosage for administration to the patient,
whether, for example, by one or more separate administrations, or
by continuous infusion. One typical daily dosage might range from
about 1 .mu.g/kg to 100 mg/kg or more, depending on the factors
mentioned above. For repeated administrations over several days or
longer, depending on the condition, the treatment would generally
be sustained until a desired suppression of disease symptoms
occurs. Such doses may be administered intermittently, e.g. every
week or every three weeks (e.g. such that the patient receives from
about two to about twenty, or e.g. about six doses of the
antibody). An initial higher loading dose, followed by one or more
lower doses may be administered. However, other dosage regimens may
be useful. The progress of this therapy is easily monitored by
conventional techniques and assays.
[0565] It is understood that any of the above formulations or
therapeutic methods may be carried out using an immunoconjugate of
the invention in place of or in addition to an anti-OX40 antibody
and/or anti-PDL1 antibody of the present disclosure.
III. Articles of Manufacture and Kits
[0566] In another aspect of the invention, an article of
manufacture or kit containing materials useful for the treatment,
prevention and/or diagnosis of the disorders described above is
provided. The article of manufacture comprises a container and a
label or package insert on or associated with the container.
Suitable containers include, for example, bottles, vials, syringes,
IV solution bags, etc. The containers may be formed from a variety
of materials such as glass or plastic. The container holds a
composition which is by itself or combined with another composition
effective for treating, preventing and/or diagnosing the condition
and may have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection needle). At least one active
agent in the composition is an antibody of the invention. The label
or package insert indicates that the composition is used for
treating the condition of choice. Moreover, the article of
manufacture may comprise (a) a first container with a composition
contained therein, wherein the composition comprises an antibody of
the invention; and (b) a second container with a composition
contained therein, wherein the composition comprises a further
cytotoxic or otherwise therapeutic agent. The article of
manufacture in this embodiment of the invention may further
comprise a package insert indicating that the compositions can be
used to treat a particular condition. Alternatively, or
additionally, the article of manufacture may further comprise a
second (or third) container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution
and dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
[0567] In some embodiments, the article of manufacture or kit
contains a container including an anti-human OX40 agonist antibody
of the present disclosure for administration at a dose described
herein, e.g., a dose selected from about 0.8 mg, about 3.2 mg,
about 12 mg, about 40 mg, about 130 mg, about 400 mg, and about
1200 mg. In some embodiments, the article of manufacture or kit
contains a container including an anti-PDL1 antibody of the present
disclosure for administration at a dose described herein, e.g., a
dose of about 1200 mg. For example, the container may contain an
amount of antibody higher than the intended dose, e.g., to account
for incomplete transfer of the antibody during administration. In
some embodiments, the article of manufacture or kit contains a
container including an anti-VEGF antibody of the present disclosure
for administration at a dose described herein, e.g., a dose of
about 15 mg/kg.
[0568] In some embodiments, provided herein is a kit comprising a
medicament comprising an anti-human OX40 agonist antibody and/or an
anti-PDL1 antibody described herein and/or an anti-VEGF antibody
described herein and an optional pharmaceutically acceptable
carrier. In some embodiments, the kit further comprises
instructions for administration of the medicament for treatment of
cancer.
[0569] It is understood that any of the above articles of
manufacture may include an immunoconjugate of the invention in
place of or in addition to an anti-OX40 antibody and/or an
anti-PDL1 antibody.
TABLE-US-00019 Sequences Name SEQUENCE SEQ ID NO: Human OX40
LHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPG 1 (lacking the
FYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAG signal peptide)
TQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHT
LQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRT
SQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRR
DQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI HVR-H1- DSYMS 2 1A7.gr.1
1A7.gr.2 1A7.gr.3 1A7.gr.4 1A7.gr.5 1A7.gr.5' 1A7.gr.6 1A7.gr.7
1A7.gr.7' 1A7.gr.NADS 1A7.gr.NADA 1A7.gr.NGDA 1A7.gr.SGDS
1A7.gr.NGSS 1A7.Ala.1 1A7.Ala.2 1A7.Ala.3 1A7.Ala.4 1A7.Ala.5
1A7.Ala.6 1A7.Ala.7 1A7.Ala.8 1A7.Ala.9 1A7.Ala.10 1A7.Ala.11
1A7.Ala.12 1A7.Ala.13 1A7.Ala.14 1A7.Ala.15 1A7.Ala.16 HVR-H2-
DMYPDNGDSSYNQKFRE 3 1A7.gr.1 1A7.gr.2 1A7.gr.3 1A7.gr.4 1A7.gr.5
1A7.gr.5' 1A7.gr.6 1A7.gr.7 1A7.gr.7' 1A7.gr.DA 1A7.gr.ES 1A7.Ala.1
1A7.Ala.2 1A7.Ala.3 1A7.Ala.4 1A7.Ala.5 1A7.Ala.6 1A7.Ala.7
1A7.Ala.8 1A7.Ala.9 1A7.Ala.10 1A7.Ala.11 1A7.Ala.12 1A7.Ala.13
1A7.Ala.14 1A7.Ala.15 1A7.Ala.16 HVR-H3- APRWYFSV 4 1A7.gr.1
1A7.gr.2 1A7.gr.3 1A7.gr.4 1A7.gr.5 1A7.gr.5' 1A7.gr.6 1A7.gr.7
1A7.gr.7' 1A7.gr.DA 1A7.gr.ES 1A7.gr.NADS 1A7.gr.NADA 1A7.gr.NGDA
1A7.gr.SGDS 1A7.gr.NGSS 1A7.gr.DANAD A 1A7.Ala.1 1A7.Ala.2
1A7.Ala.3 1A7.Ala.4 1A7.Ala.5 1A7.Ala.6 1A7.Ala.7 1A7-Ala.15
1A7.A1a.16 HVR-L1- RASQDISNYLN 5 1A7.gr.1 1A7.gr.2 1A7.gr.3
1A7.gr.4 1A7.gr.5 1A7.gr.5' 1A7.gr.6 1A7.gr.7 1A7.gr.7' 1A7.gr.DA
1A7.gr.ES 1A7.gr.NADS 1A7.gr.NADA 1A7.gr.NGDA 1A7.gr.SGDS
1A7.gr.NGSS 1A7.gr.DANAD A 1A7.Ala.1 1A7.Ala.2 1A7.Ala.3 1A7.Ala.4
1A7.Ala.5 1A7.Ala.6 1A7.Ala.7 1A7.Ala.8 1A7.Ala.9 1A7.Ala.10
1A7.Ala.11 1A7.Ala.12 1A7.Ala.13 1A7.Ala.14 1A7.Ala.15 1A7.Ala.16
HVR-L2- YTSRLRS 6 1A7.gr.1 1A7.gr.2 1A7.gr.3 1A7.gr.4 1A7.gr.5
1A7.gr.5' 1A7.gr.6 1A7.gr.7 1A7.gr.7' 1A7.gr.DA 1A7.gr.ES
1A7.gr.NADS 1A7.gr.NADA 1A7.gr.NGDA 1A7.gr.SGDS 1A7.gr.NGSS
1A7.gr.DANAD A 1A7.Ala.1 1A7.Ala.2 1A7.Ala.3 1A7.Ala.4 1A7.Ala.5
1A7.Ala.6 1A7.Ala.7 1A7.Ala.8 1A7.Ala.9 1A7.Ala.10 1A7.Ala.11
1A7.Ala.12 1A7.Ala.13 1A7.A1a.14 1A7.Ala.15 1A7.Ala.16 HVR-L3-
QQGHTLPPT 7 1A7.gr.1 1A7.gr.2 1A7.gr.3 1A7.gr.4 1A7.gr.5 1A7.gr.5'
1A7.gr.6 1A7.gr.7 1A7.gr.7' 1A7.gr.DA 1A7.gr.ES 1A7.gr.NADS
1A7.gr.NADA 1A7.gr.NGDA 1A7.gr.SGDS 1A7.gr.NGSS 1A7.gr.DANAD A
1A7.Ala.8 1A7.Ala.9 1A7.Ala.10 1A7.Ala.11 1A7.Ala.12 1A7.Ala.13
1A7.Ala.14 1A7.Ala.15 1A7.Ala.16 HVR-H1- DAYMS 8 1A7.gr.DA HVR-H1-
ESYMS 9 1A7.gr.ES 1A7.gr.DANAD A HVR-H2- DMYPDNADSSYNQKFRE 10
1A7.gr.NADS HVR-H2- DMYPDNADASYNQKFRE 11 1A7.gr.NADA 1A7.gr.DANAD A
HVR-H2- DMYPDNGDASYNQKFRE 12 1A7.gr.NGDA HVR-H2- DMYPDSGDSSYNQKFRE
13 1A7.gr.SGDS HVR-H2- DMYPDNGSSSYNQKFRE 14 1A7.gr.NGSS HVR-H3-
APRWYFSA 15 1A7.Ala.8 HVR-H3- APRWYASV 16 1A7.Ala.9 HVR-H3-
APRWAFSV 17 1A7.Ala.10 HVR-H3- APAWYFSV 18 1A7.Ala.11 HVR-H3-
APRWYFAV 19 1A7.Ala.12 HVR-H3- APRAYFSV 20 1A7.Ala.13 HVR-H3-
AARWYFSV 21 1A7.Ala.14 HVR-L3- QQGHTLPAT 22 1A7.Ala.1
HVR-L3- QQGHTAPPT 23 1A7.Ala.2 HVR-L3- QQGATLPPT 24 1A7.Ala.3
HVR-L3- QQGHALPPT 25 1A7.Ala.4 HVR-L3- QQAHTLPPT 26 1A7.Ala.5
HVR-L3- QQGHTLAPT 27 1A7.Ala.6 HVR-L3- QAGHTLPPT 28 1A7.Ala.7
HVR-H1- NYLIE 29 3C8.gr.1 3C8.gr.2 3C8.gr.3 3C8.gr.4 3C8.gr.5
3C8.gr.5.SG 3C8.gr.5.EG 3C8.gr.5.QG 3C9.gr.5.DQ 3C8.gr.5.DA
3C8.gr.6 3C8.gr.7 3C8.gr.8 3C8.gr.9 3C8.gr.10 3C8.gr.11 3C8.A.1
3C8.A.2 3C8.A.3 3C8.A.4 3C8.A.5 3C8.A.6 3C8.A.7 3C8.A.8 3C8.A.9
3C8.A.10 HVR-H2- VINPGSGDTYYSEKFKG 30 3C8.gr.1 3C8.gr.2 3C8.gr.3
3C8.gr.4 3C8.gr.5 3C8.gr.5.SG 3C8.gr.5.EG 3C8.gr.5.QG 3C8.gr.6
3C8.gr.7 3C8.gr.8 3C8.gr.9 3C8.gr.10 3C8.gr.11 3C8.A.1 3C8.A.2
3C8.A.3 3C8.A.4 3C8.A.5 3C8.A.6 3C8.A.7 3C8.A.8 3C8.A.9 3C8.A.10
HVR-H2- VINPGSGDAYYSEKFKG 31 3C8.gr.5.DA HVR-H2- VINPGSGDQYYSEKFKG
32 3C8.gr.5.DQ HVR-H3- DRLDY 33 3C8.gr.1 3C8.gr.2 3C8.gr.3 3C8.gr.4
3C8.gr.5 3C8.gr.5.SG 3C8.gr.5.EG 3C8.gr.5.QG 3C8.gr.5.DA
3C8.gr.5.DQ 3C8.gr.6 3C8.gr.7 3C8.gr.8 3C8.gr.9 3C8.gr.10 3C8.gr.11
3C8.A.1 3C8.A.2 3C8.A.3 3C8.A.4 3C8.A.5 3C8.A.6 3C8.A.7 HVR-H3-
ARLDY 34 3C8.A.8 HVR-H3- DALDY 35 3C8.A.9 HVR-H3- DRADY 36 3C8.A.10
HVR-L1- HASQDISSYIV 37 3C8.gr.1 3C8.gr.2 3C8.gr.3 3C8.gr.4 3C8.gr.5
3C8.gr.5.SG 3C8.gr.5.EG 3C8.gr.5.QG 3C8.gr.5.DA 3C8.gr.5.DQ
3C8.gr.6 3C8.gr.7 3C8.gr.8 3C8.gr.9 3C8.gr.10 3C8.gr.11 3C8.A.1
3C8.A.2 3C8.A.3 3C8.A.4 3C8.A.5 3C8.A.6 3C8.A.7 3C8.A.8 3C8.A.9
3C8.A.10 HVR-L2- HGTNLED 38 3C8.gr.1 3C8.gr.2 3C8.gr.3 3C8.gr.4
3C8.gr.5 3C8.gr.5.DA 3C8.gr.5.DQ 3C8.gr.6 3C8.gr.7 3C8.gr.8
3C8.gr.9 3C8.gr.10 3C8.gr.11 3C8.A.1 3C8.A.2 3C8.A.3 3C8.A.4
3C8.A.5 3C8.A.6 3C8.A.7 3C8.A.8 3C8.A.9 3C8.A.10 HVR-L2- HGTNLES 39
3C8.gr5.SG HVR-L2- HGTNLEE 40 3C8.gr.5.EG HVR-L2- HGTNLEQ 41
3C8.gr.5.QG HVR-L3 VHYAQFPYT 42 3C8.gr.1 3C8.gr.2 3C8.gr.3 3C8.gr.4
3C8.gr.5 3C8.gr.5.SG 3C8.gr.5.EG 3C8.gr.5.QG 3C8.gr.5.DA
3C8.gr.5.DQ 3C8.gr.6 3C8.gr.7 3C8.gr.8 3C8.gr.9 3C8.gr.10 3C8.gr.11
3C8.A.8 3C8.A.9 3C8.A.10 HVR-L3- AHYAQFPYT 43 3C8.A.1 HVR-L3-
VAYAQFPYT 44 3C8.A.2 HVR-L3- VHAAQFPYT 45 3C8.A.3 HVR-L3- VHYAAFPYT
46 3C8.A.4 HVR-L3- VHYAQAPYT 47 3C8.A.5 HVR-L3- VHYAQFAYT 48
3C8.A.6 HVR-L3- VHYAQFPAT 49 3C8.A.7 HVR-H1- DYGVL 50 1D2.gr.1
1D2.gr.2 1D2.gr.3 HVR-H2- MIWSGGTTDYNAAFIS 51 1D2.gr.1 1D2.gr.2
1D2.gr.3 HVR-H3- EEMDY 52 1D2.gr.1 1D2.gr.2 1D2.gr.3 HVR-L1-
RASQDISNFLN 53 1D2.gr.1 1D2.gr.2 1D2.gr.3 HVR-L2- YTSRLHS 54
1D2.gr.1 1D2.gr.2 1D2.gr.3 HVR-L3- QQGNTLPWT 55 1D2.gr.1 1D2.gr.2
1D2.gr.3 1A7.gr.1 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 56
V.sub.H GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELS
SLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS
1A7.gr.1 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 57 V.sub.L
PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK
1A7.gr.2 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 58 V.sub.H
GQGLEWIGDMYPDNGDSSYNQKFRERVTITVDTSTSTAYLELS
SLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.gr.2
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 59 V.sub.L
PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK
1A7.gr.3 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 60 V.sub.H
GQGLEWIGDMYPDNGDSSYNQKFRERVTLTVDTSTSTAYLEL
SSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.gr.3
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 61 V.sub.L
PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK
1A7.gr.4 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 62 V.sub.H
GQGLEWIGDMYPDNGDSSYNQKFRERVTITVDTSTSTAYLELS
SLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.gr.4
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKT 63 V.sub.L
VKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK
1A7.gr.5 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 64 V.sub.H
GQGLEWIGDMYPDNGDSSYNQKFRERVTITVDTSTSTAYLELS
SLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.gr.5
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKT 65 V.sub.L
VKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK
1A7.gr.6 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 66 V.sub.H
GQGLEWIGDMYPDNGDSSYNQKFRERVTITVDTSTSTAYLELS
SLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.gr.6
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKT 67 V.sub.L
VKLLIYYTSRLRSGVPSRFSGSGSGKDYTLTISSLQPEDFATYFC QQGHTLPPTFGQGTKVEIK
1A7.gr.7 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 68 V.sub.H
GQGLEWIGDMYPDNGDSSYNQKFRERVTITVDTSTSTAYLELS
SLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.gr.7
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKT 69 V.sub.L
VKLLIYYTSRLRSGVPSRFSGSGSGKDYTLTISSLQPEDFATYFC QQGHTLPPTFGQGTKVEIK
1A7.gr.DA EVQLVQSGAEVKKPGASVKVSCKASGYTFTDAYMSWVRQAP 70 V.sub.H
GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELS
SLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.gr.DA
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 71 V.sub.L
PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK
1A7.gr.ES EVQLVQSGAEVKKPGASVKVSCKASGYTFTESYMSWVRQAP 72 V.sub.H
GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELS
SLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.gr.ES
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 73 V.sub.L
PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK
1A7.gr.NADS EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 74 V.sub.H
GQGLEWIGDMYPDNADSSYNQKFRERVTITRDTSTSTAYLELS
SLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.gr.NADS
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 75 V.sub.L
PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK
1A7.gr.NADA EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 76 V.sub.H
GQGLEWIGDMYPDNADASYNQKFRERVTITRDTSTSTAYLELS
SLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.gr.NADA
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 77 V.sub.L
PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK
1A7.gr.NGDA EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 78 V.sub.H
GQGLEWIGDMYPDNGDASYNQKFRERVTITRDTSTSTAYLELS
SLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.gr.NGDA
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 79 V.sub.L
PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK
1A7.gr.SGDS EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 80 V.sub.H
GQGLEWIGDMYPDSGDSSYNQKFRERVTITRDTSTSTAYLELS
SLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.gr.SGDS
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 81 V.sub.L
PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK
1A7.gr.NGSS EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 82 V.sub.H
GQGLEWIGDMYPDNGSSSYNQKFRERVTITRDTSTSTAYLELS
SLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.gr.NGSS
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 83 V.sub.L
PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK
1A7.gr.DANADA EVQLVQSGAEVKKPGASVKVSCKASGYTFTDAYMSWVRQAP 84 V.sub.H
GQGLEWIGDMYPDNADASYNQKFRERVTITRDTSTSTAYLELS
SLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.gr.DANADA
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 85 V.sub.L
PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK
1A7.Ala.1 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 86 V.sub.H
GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELS
SLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.Ala.1
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 87 V.sub.L
PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPATFGQGTKVEIK
1A7.Ala.2 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 88 V.sub.H
GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELS
SLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.Ala.2
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 89 V.sub.L
PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTAPPTFGQGTKVEIK
1A7.Ala.3 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 90 V.sub.H
GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELS
SLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.Ala.3
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 91 V.sub.L
PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGATLPPTFGQGTKVEIK
1A7.Ala.4 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 92 V.sub.H
GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELS
SLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.Ala.4
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 93 V.sub.L
PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHALPPTFGQGTKVEIK
1A7.Ala.5 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 94 V.sub.H
GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELS
SLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.Ala.5
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 95 V.sub.L
PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQAHTLPPTFGQGTKVEIK
1A7.Ala.6 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 96 V.sub.H
GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELS
SLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.Ala.6
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 97 V.sub.L
PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLAPTFGQGTKVEIK
1A7.Ala.7 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 98 V.sub.H
GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELS
SLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.Ala.7
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 99 V.sub.L
PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QAGHTLPPTFGQGTKVEIK
1A7.Ala.8 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 100 V.sub.H
GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELS
SLRSEDTAVYYCVLAPRWYFSAWGQGTLVTVSS 1A7.Ala.8
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 101 V.sub.L
PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK
1A7.Ala.9 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 102 V.sub.H
GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELS
SLRSEDTAVYYCVLAPRWYASVWGQGTLVTVSS 1A7.Ala.9
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 103 V.sub.L
PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK
1A7.Ala.10 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 104 V.sub.H
GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELS
SLRSEDTAVYYCVLAPRWAFSVWGQGTLVTVSS 1A7.Ala.10
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 105 V.sub.L
PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK
1A7.Ala.11 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 106 V.sub.H
GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELS
SLRSEDTAVYYCVLAPAWYFSVWGQGTLVTVSS 1A7.Ala.11
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 107 V.sub.L
PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK
1A7.Ala.12 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 108 V.sub.H
GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELS
SLRSEDTAVYYCVLAPRWYFAVWGQGTLVTVSS 1A7.Ala.12
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 109 V.sub.L
PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK
1A7.Ala.13 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 110 V.sub.H
GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELS
SLRSEDTAVYYCVLAPRAYFSVWGQGTLVTVSS 1A7.Ala.13
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 111 V.sub.L
PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK
1A7.Ala.14 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 112 V.sub.H
GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELS
SLRSEDTAVYYCVLAARWYFSVWGQGTLVTVSS 1A7.Ala.14
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 113 V.sub.L
PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK
1A7.Ala.15 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 114 V.sub.H
GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELS
SLRSEDTAVYYCALAPRWYFSVWGQGTLVTVSS 1A7.Ala.15
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 115 V.sub.L
PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK
1A7.Ala.16 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 116 V.sub.H
GQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELS
SLRSEDTAVYYCVAAPRWYFSVWGQGTLVTVSS 1A7.Ala.16
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKA 117 V.sub.L
PKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGHTLPPTFGQGTKVEIK
3C8.gr.1 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 118 V.sub.H
QGLEWIGVINPGSGDTYYSEKFKGRVTITRDTSTSTAYLELSSL
RSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.gr.1
DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKAP 119 V.sub.L
KLLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
VHYAQFPYTFGQGTKVEIK
3C8.gr.2 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 120 V.sub.H
QGLEWIGVINPGSGDTYYSEKFKGRVTITADTSTSTAYLELSSL
RSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.gr.2
DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKAP 121 V.sub.L
KLLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQFPYTFGQGTKVEIK
3C8.gr.3 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 122 V.sub.H
QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSL
RSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.gr.3
DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKAP 123 V.sub.L
KLLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQFPYTFGQGTKVEIK
3C8.gr.4 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 124 V.sub.H
QGLEWIGVINPGSGDTYYSEKFKGRVTITADTSTSTAYLELSSL
RSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.gr.4
DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 125 V.sub.L
KGLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQFPYTFGQGTKVEIK
3C8.gr.5 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 126 V.sub.H
QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSL
RSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.gr.5
DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 127 V.sub.L
KGLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQFPYTFGQGTKVEIK
3C8.gr.5.SG EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 128 V.sub.H
QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSL
RSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.gr.5.SG
DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 129 V.sub.L
KGLIYHGTNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCV HYAQFPYTFGQGTKVEIK
3C8.gr.5.EG EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 130 V.sub.H
QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSL
RSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.gr.5.EG
DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 131 V.sub.L
KGLIYHGTNLEEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQFPYTFGQGTKVEIK
3C8.gr.5.QG EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 132 V.sub.H
QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSL
RSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.gr.5.QG
DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 133 V.sub.L
KGLIYHGTNLEQGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQFPYTFGQGTKVEIK
3C8.gr.6 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 134 V.sub.H
QGLEWIGVINPGSGDTYYSEKFKGRVTITADTSTSTAYLELSSL
RSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.gr.6
DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 135 V.sub.L
KGLIYHGTNLEDGVPSRFSGSGSGADYTLTISSLQPEDFATYYC VHYAQFPYTFGQGTKVEIK
3C8.gr.7 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 136 V.sub.H
QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSL
RSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.gr.7
DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 137 V.sub.L
KGLIYHGTNLEDGVPSRFSGSGSGADYTLTISSLQPEDFATYYC VHYAQFPYTFGQGTKVEIK
3C8.gr.8 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 138 V.sub.H
QGLEWIGVINPGSGDTYYSEKFKGRVTLTRDTSTSTAYLELSSL
RSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.gr.8
DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 139 V.sub.L
KGLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQFPYTFGQGTKVEIK
3C8.gr.9 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 140 V.sub.H
QGLEWIGVINPGSGDTYYSEKFKGRVTLTRDTSTSTAYLELSSL
RSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.gr.9
DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSP 141 V.sub.L
KLLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQFPYTFGQGTKVEIK
3C8.gr.10 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 142 V.sub.H
QGLEWIGVINPGSGDTYYSEKFKGRVTLTRDTSTSTAYLELSSL
RSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.gr.10
DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKAF 143 V.sub.L
KLLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQFPYTFGQGTKVEIK
3C8.gr.11 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 144 V.sub.H
QGLEWIGVINPGSGDTYYSEKFKGRVTLTRDTSTSTAYLELSSL
RSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.gr.11
DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKAP 145 V.sub.L
KGLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQFPYTFGQGTKVEIK
3C8.A.1 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 146 V.sub.H
QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSL
RSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.A.1
DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 147 V.sub.L
KGLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC AHYAQFPYTFGQGTKVEIK
3C8.A.2 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 148 V.sub.H
QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSL
RSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.A.2
DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 149 V.sub.L
KGLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VAYAQFPYTFGQGTKVEIK
3C8.A.3 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 150 V.sub.H
QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSL
RSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.A.3
DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 151 V.sub.L
KGLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHAAQFPYTFGQGTKVEIK
3C8.A.4 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 152 V.sub.H
QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSL
RSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.A.4
DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 153 V.sub.L
KGLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAAFPYTFGQGTKVEIK
3C8.A.5 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 154 V.sub.H
QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSL
RSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.A.5
DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 155 V.sub.L
KGLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQAPYTFGQGTKVEIK
3C8.A.6 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 156 V.sub.H
QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSL
RSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.A.6
DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 157 V.sub.L
KGLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQFAYTFGQGTKVEIK
3C8.A.7 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 158 V.sub.H
QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSL
RSEDTAVYYCARDRLDYWGQGTLVTVSS 3C8.A.7
DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 159 V.sub.L
KGLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQFPATFGQGTKVEIK
3C8.A.8 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 160 V.sub.H
QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSL
RSEDTAVYYCARARLDYWGQGTLVTVSS 3C8.A.8
DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 161 V.sub.L
KGLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQFPYTFGQGTKVEIK
3C8.A.9 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 162 V.sub.H
QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSL
RSEDTAVYYCARDALDYWGQGTLVTVSS 3C8.A.9
DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 163 V.sub.L
KGLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQFPYTFGQGTKVEIK
3C8.A.10 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNYLIEWVRQAPG 164 V.sub.H
QGLEWIGVINPGSGDTYYSEKFKGRVTLTADTSTSTAYLELSSL
RSEDTAVYYCARDRADYWGQGTLVTVSS 3C8.A.10
DIQMTQSPSSLSASVGDRVTITCHASQDISSYIVWYQQKPGKSF 165 V.sub.L
KGLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC VHYAQFPYTFGQGTKVEIK
1D2.gr.1 EVQLVESGPGLVKPSETLSLTCTVSGFSLTDYGVLWIRQPPGKG 166 V.sub.H
LEWIGMIWSGGTTDYNAAFISRVTISVDTSKNQFSLKLSSVTAA
DTAVYYCVREEMDYWGQGTLVTVSS 1D2.gr.1
DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGKA 167 V.sub.L
PKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGNTLPWTFGQGTKVEIK
1D2.gr.2 EVQLVESGPGLVKPSETLSLTCTVSGFSLTDYGVLWIRQPPGKG 168 V.sub.H
LEWIGMIWSGGTTDYNAAFISRVTISKDTSKNQVSLKLSSVTA
ADTAVYYCVREEMDYWGQGTLVTVSS 1D2.gr.2
DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGKA 169 V.sub.L
PKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGNTLPWTFGQGTKVEIK
1D2.gr.3 EVQLVESGPGLVKPSETLSLTCTVSGFSLTDYGVLWVRQPPGK 170 V.sub.H
GLEWLGMIWSGGTTDYNAAFISRLTISKDTSKNQVSLKLSSVT
AADTAVYYCVREEMDYWGQGTLVTVSS 1D2.gr.3
DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGKA 171 V.sub.L
PKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGNTLPWTFGQGTKVEIK
CON1 X.sub.1X.sub.2YMS, wherein X.sub.1 is D or E, and X.sub.2 is S
172 (1A7)HVR-H1 or A CON1 (1A7)
DMYPDX.sub.1X.sub.2X.sub.3X.sub.4SYNQKFRE, wherein X.sub.1 is N or
S, X.sub.2 173 HVR-H2 is A or G, X.sub.3 iS D or S, and X.sub.4 iS
A or S CON1 (1A7) APRWX.sub.1X.sub.2X.sub.3X.sub.4, wherein X.sub.1
is Y or A, X.sub.2 is A or 174 HVR-H3 F, X.sub.3 is S or A, and
X.sub.4 is A or V. CON1 (1A7)
QX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7T, wherein
X.sub.1 is A or Q, X.sub.2 is A 175 HVR-L3 or G, X.sub.3 is A or H,
X.sub.4 is A or T, X.sub.5 is A or L, X.sub.6 is A or P, and
X.sub.7 is A or P. CON2 (3C8) VINPGSGDX.sub.1YYSEKFKG, wherein
X.sub.1 is T, A or Q. 176 HVR-H2 CON2 (3C8) HGTNLEX.sub.1, wherein
X.sub.1 is S, E, or Q. 177 HVR-L2 CON2 (3C8)
X.sub.1X.sub.2YAQFPYX.sub.3, wherein X.sub.1 is V or A, X.sub.2 is
H 178 HVR-L3 or A, and X.sub.3 is Y or A. 1A7 V.sub.L
DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGT 179
VKLLIYYTSRLRSGVPSRFSGSGSGKDYFLTISNLEQEDVAAYF CQQGHTLPPTFGGGTKLEIK
1A7 V.sub.H EVQLQQSGPELVKPGASVKISCKASGYTFTDSYMSWVKQSHG 180
KTLEWIGDMYPDNGDSSYNQKFREKVTLTVDKSSTTAYMEFR
SLTSEDSAVYYCVLAPRWYFSVWGTGTTVTVSS 3C8 V.sub.L
DILMTQSPSSMSVSLGDTVSITCHASQDISSYIVWLQQKPGKSF 181
RGLIYHGTNLEDGIPSRFSGSGSGADYSLTISSLESEDFADYYCV HYAQFPYTFGGGTKLEIK
3C8 V.sub.H QVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLIEWVKQRPG 182
QGLEWIGVINPGSGDTYYSEKFKGKVTLTADKSSSTAYMQLSS
LTSEDSAVYFCARDRLDYWGQGTTLTVSS
1A7.gr.5' EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 183 V.sub.H
GQGLEWIGDMYPDNGDSSYNQKFRERVTLTVDTSTSTAYLEL
SSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 1A7.gr.7'
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAP 184 V.sub.H
GQGLEWIGDMYPDNGDSSYNQKFRERVTLTVDTSTSTAYLEL
SSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS
EXAMPLES
Example 1
A Phase I Dose Escalation Study of the Safety and Pharmacokinetics
of MOXR0916 and MPDL3280A in Patients with Locally Advanced or
Metastatic Solid Tumors
Study Design
[0570] This is a Phase Ib, open-label, multicenter, dose-escalation
study designed to evaluate the safety, tolerability, and
pharmacokinetics of the combination of MOXR0916 (1A7.gr1 IgG1) and
MPDL3280A in patients with locally advanced, recurrent, or
metastatic incurable solid malignancy that has progressed after
available standard therapy; or for which standard therapy has
proven to be ineffective or intolerable, or is considered
inappropriate; or for which a clinical trial of an investigational
agent is a recognized standard of care. Approximately 184-360
patients may be enrolled in this study at approximately 30 study
centers globally.
[0571] This study includes a screening period, a treatment period,
and a post-treatment follow-up period. Patients may be enrolled in
two stages: a dose-escalation stage and an expansion stage (FIG.
1).
[0572] As described in greater detail below, MOXR0916 and MPDL3280A
are each administered by intravenous (IV) infusion on Day 1 of
21-day cycles. In the absence of unacceptable toxicity or
clinically compelling disease progression, treatment with both
agents may be continued beyond Cycle 1 based on a favorable
assessment of benefit and risk by the investigator.
[0573] All adverse events (AEs) may be monitored and recorded for
at least 90 days after the last dose of study treatment or until
initiation of another systemic anti-cancer therapy, whichever
occurs first. After this period, the Sponsor should be notified if
the investigator becomes aware of any serious adverse events if the
event is believed to be related to prior study drug treatment.
Adverse events may be graded according to National Cancer Institute
Common Terminology Criteria for Adverse Events Version 4 (NCI CTCAE
v4.0).
[0574] To characterize the pharmacokinetic (PK) properties of
MOXR0916 and MPDL3280A and pharmacodynamic responses to treatment,
blood samples are taken at various timepoints before and after
dosing. Patients undergo tumor assessments at screening and during
the study. Patients may be permitted to continue study treatment
even if standard RECIST v1.1 criteria for progressive disease are
met, provided they meet the criteria for continued treatment. All
patients who discontinue MOXR0916 and MPDL3280A for reasons other
than disease progression (e.g., adverse events) continue tumor
assessments. Patients who discontinue MOXR0916 and MPDL3280A may
return to the clinic for a treatment discontinuation visit within
30 days after the last dose of study treatment. All patients may be
followed for survival and subsequent anti-cancer therapy
information approximately every 3 months until death, loss to
follow-up, or study termination, unless the patient requests to be
withdrawn from follow-up.
Study Objectives
[0575] The primary objective for this study is to evaluate the
safety and tolerability of the combination of MOXR0916 and
MPDL3280A in patients with locally advanced or metastatic solid
tumors.
[0576] The secondary objectives for this study are as follows:
(a) To estimate the maximum tolerated dose (MTD) of MOXR0916 when
administered in combination with MPDL3280A and to characterize the
dose-limiting toxicities (DLTs); (b) To identify a recommended
Phase II dose for MOXR0916 administered in combination with
MPDL3280A; (c) To characterize the pharmacokinetics of MOXR0916 and
MPDL3280A when administered in combination; (d) To characterize the
immunogenic potential of MOXR0916 and MPDL3280A when administered
in combination by measuring anti-MOXR0916 and anti-MPDL3280A
antibodies, respectively, and assessing their relationship with
other outcome measures; and (e) To make a preliminary assessment of
the anti-tumor activity of the combination of MOXR0916 and
MPDL3280A in patients with locally advanced or metastatic solid
tumors.
[0577] The exploratory objectives for this study are as
follows:
(a) To make a preliminary assessment of biomarkers that might act
as pharmacodynamic indicators of activity of the combination of
MOXR0916 and MPDL3280A in patients with locally advanced or
metastatic solid tumors; and (b) To make a preliminary assessment
of biomarkers that might act as predictors of anti-tumor activity
of the combination of MOXR0916 and MPDL3280A in patients with
locally advanced or metastatic solid tumors.
Study Population
[0578] Patients must meet the following criteria for study entry,
which include cancer-specific (both general and specific for the
dose-expansion stage) and general inclusion criteria.
[0579] Cancer-specific inclusion criteria include the
following:
(a) Histologic documentation of locally advanced, recurrent or
metastatic incurable solid malignancy that has progressed after
available standard therapy; or for which standard therapy has
proven to be ineffective or intolerable, or is considered
inappropriate; or for which a clinical trial of an investigational
agent is a recognized standard of care; (b) Confirmed availability
of representative tumor specimens in paraffin blocks (preferred) or
.gtoreq.15 unstained slides, with an associated pathology report.
Acceptable samples include core needle biopsies for deep tumor
tissue (minimum three cores) or excisional, incisional, punch, or
forceps biopsies for cutaneous, subcutaneous, or mucosal lesions.
Fine-needle aspiration, brushing, cell pellet from effusions or
ascites, and lavage samples are not acceptable. Tumor tissue from
bone metastases is not evaluable for PD-L1 expression and is
therefore not acceptable. If adequate tissue from distinct time
points (such as time of initial diagnosis and time of disease
recurrence) and/or multiple metastatic tumors is available,
priority should be given to the tissue most recently collected
(ideally subsequent to the most recent systemic therapy). Multiple
samples may be collected for a given patient, on the basis of
availability; however, the requirement for a block or .gtoreq.15
unstained slides should be satisfied by a single biopsy or
resection specimen. Prior to signing the main study informed
consent form, patients may sign a pre-screening consent form to
specifically allow the collection and testing of archival or fresh
tumor specimens. A patient with insufficient or unavailable
archival tissue may be eligible, upon discussion with the Medical
Monitor, if the patient meets any of the following: Can provide at
least 10 unstained, serial slides; Is willing to consent to and
undergo a pretreatment core, punch, or excisional/incisional biopsy
sample collection of the tumor; or Is to be enrolled in a
dose-escalation cohort; and (c) Measurable disease per RECIST v1.1
(for RECIST v1.1 criteria and additional descriptions related to
measurements of tumors and tumor response, see, e.g., Eisenhauer,
E. A. et al. (2009) Eur. J. Cancer 45:228-247).
[0580] In some embodiments, modified RECIST criteria may be used to
evaluate tumor response. Modified Response Evaluation Criteria in
Solid Tumors (RECIST) is derived from RECIST, Version 1.1 (v1.1)
conventions (see, e.g., Eisenhauer, E. A. et al. (2009) Eur. J.
Cancer 45:228-247) and immune-related response criteria (irRC; see,
e.g., Wolchok et al. (2009) Clin. Can. Res. 15:7412-7420; Nishino
et al. (2014) J. Immunother. Can. 2:17; and Nishino et al. (2013)
Clin. Can. Res. 19:3936-3943). Conventional response criteria may
not be adequate to characterize the anti-tumor activity of
immunotherapeutic agents like MPDL3280A, which can produce delayed
responses that may be preceded by initial apparent radiographic
progression, including the appearance of new lesions. Therefore,
modified response criteria have been developed that account for the
possible appearance of new lesions and allow radiological
progression to be confirmed at a subsequent assessment. For a
summary of the changes between modified RECIST and RECIST v1.1, see
Table B above.
[0581] When not otherwise specified, RECIST v1.1 conventions apply.
Briefly, modified RECIST criteria for determining objective tumor
response for target lesions include:
(a) Complete response (CR): disappearance of all target and
non-target lesions. Lymph nodes that shrink to <10 mm short axis
are considered normal; (b) Partial response (PR): at least a 30%
decrease in the sum of the diameters of all target and all new
measurable lesions, taking as reference the baseline sum of
diameters, in the absence of CR. Note: the appearance of new
measurable lesions is factored into the overall tumor burden, but
does not automatically qualify as progressive disease until the sum
of the diameters increases by .gtoreq.20% when compared with the
sum of the diameters at nadir; (c) Progressive disease (PD): at
least a 20% increase in the sum of diameters of all target and
selected new measurable lesions, taking as reference the smallest
sum on study (nadir SLD; this includes the baseline sum if that is
the smallest on study). In addition to the relative increase of
20%, the sum must also demonstrate an absolute increase of at least
5 mm; and (d) Stable disease (SD): neither sufficient shrinkage to
qualify for PR nor sufficient increase to qualify for PD, taking as
reference the smallest sum of the diameters while on study.
[0582] The assessment of non-target lesions may be captured on the
CRF at each timepoint using standard RECIST v1.1 definitions of CR,
non-CR/non-PD, and PD (unequivocal progression). However, in
determining the overall modified RECIST tumor response, non-target
lesions contribute only to the assessment of a complete response.
Non-target lesions are not considered in the overall definition of
PR, SC, or PD per modified RECIST.
[0583] New lesions alone do not qualify as progressive disease.
However, their contribution to total tumor burden is included in
the sum of the diameters, which is used to determine the overall
modified RECIST tumor response.
[0584] Cancer-specific inclusion criteria unique to patients in the
dose-expansion stage include the following:
(a) Expansion Part I biopsy cohort: Accessible lesion(s) that
permit a total of at least two biopsies (pretreatment and
on-treatment) without unacceptable risk of a significant procedural
complication. Acceptable samples include core needle biopsies for
deep tumor tissue or lymph nodes or excisional, incisional, punch,
or forceps biopsies for cutaneous, subcutaneous, or mucosal
lesions. Fine needle aspirates, cell pellets from effusions or
ascites, lavage samples, and bone biopsies are not permitted.
Target lesions considered for core needle biopsies should be deemed
suitable for retrieval of at least three cores at a given timepoint
(minimum diameter 18-gauge). If multiple lesions are available, it
is preferable to obtain the on-treatment biopsy from the same
lesion (or organ) as the pretreatment biopsy, if feasible, to avoid
introduction of heterogeneity related to site of metastasis; (b)
Expansion Part II biopsy cohort: Cutaneous or subcutaneous tumors
.gtoreq.5 mm in diameter amenable to serial biopsy (pretreatment
and on-treatment) by excisional, incisional or punch biopsies
without unacceptable risk of a major procedural complication. If
more than one biopsy is planned to be taken from one lesion, the
lesion must be large enough to permit successive biopsies .gtoreq.1
cm apart. If multiple lesions are available, it is preferable to
obtain the on-treatment biopsy from the same lesion (or organ) as
the pretreatment biopsy, if feasible, to avoid introduction of
heterogeneity related to site of metastasis; (c) Melanoma cohort:
Histologically confirmed incurable, advanced metastatic melanoma
(patients whose tumors have a known BRAF V600 mutation must also
have experienced disease progression, during or after treatment, or
intolerance to treatment with a BRAF and/or mitogen-activated
protein kinase kinase (MEK) kinase inhibitor); (d) RCC cohort:
Histologically confirmed incurable, advanced RCC with component of
clear cell histology and/or component of sarcomatoid histology; (e)
TNBC cohort: Histologically confirmed incurable, advanced estrogen
receptor-, progesterone receptor-, and human epidermal growth
factor receptor 2 (HER2)-negative (triple-negative) adenocarcinoma
of the breast, as defined by the American Society of Clinical
Oncology College of American Pathologists (ASCO-CAP) guidelines:
[0585] (i) <1% of tumor cell nuclei are immunoreactive for
estrogen receptor and <1% of tumor cell nuclei are
immunoreactive for progesterone receptor (Hammond, M. E. et al.
(2010) J. Clin. Oncol. 28:2784-2795) AND [0586] (ii) HER2 tests
demonstrate either immunohistochemistry (IHC) 1+, IHC 0 or in situ
hybridization (ISH) negative (Wolff, A. C. et al. (2013) J. Clin.
Oncol. 31:3997:4013); Note: Submitted archival tumor tissue must be
evaluated for PD-L1 expression prior to enrollment. Patients whose
tumor tissue is not evaluable for PD-L1 expression are not
eligible. If multiple tumor specimens are submitted (e.g., an
archival specimen and tissue from relapsed disease), patients may
be eligible if at least one specimen is evaluable for PD-L1. In the
event that enrollment (i.e., beyond approximately 20 patients) is
limited to PD-L1 selected patients, the PD-L1 score of the patient
may be the maximum PD-L1 score among the samples; (f) NSCLC cohort:
Histologically confirmed incurable, advanced NSCLC: [0587] (i)
Patients whose tumors have a known sensitizing epidermal growth
factor receptor (EGFR) mutation must also have experienced disease
progression (during or after treatment) or intolerance to treatment
with an EGFR tyrosine kinase inhibitor; [0588] (ii) Patients whose
tumors have a known anaplastic lymphoma kinase (ALK) rearrangement
must also have experienced disease progression (during or after
treatment) or intolerance to treatment with an ALK tyrosine kinase
inhibitor; Note: Submitted archival tumor tissue must be evaluated
for PD-L1 expression prior to enrollment. Patients whose tumor
tissue is not evaluable for PD-L1 expression are not eligible. If
multiple tumor specimens are submitted (e.g., an archival specimen
and tissue from relapsed disease), patients may be eligible if at
least one specimen is evaluable for PD-L1. In the event that
enrollment (i.e., beyond approximately 20 patients) is limited to
PD-L1 selected patients, the PD-L1 score of the patient may be the
maximum PD-L1 score among the samples; (g) UBC cohort:
Histologically confirmed incurable, advanced transitional cell
carcinoma of the urothelium (including renal pelvis, ureters,
urinary bladder, urethra) (Patients with mixed histologies are
required to have a dominant transitional cell pattern). Note:
Submitted archival tumor tissue must be evaluated for PD-L1
expression prior to enrollment. Patients whose tumor tissue is not
evaluable for PD-L1 expression are not eligible. If multiple tumor
specimens are submitted (e.g., an archival specimen and tissue from
relapsed disease), patients may be eligible if at least one
specimen is evaluable for PD-L1. In the event that enrollment
(i.e., beyond approximately 20 patients) is limited to PD-L1
selected patients, the PD-L1 score of the patient may be the
maximum PD-L1 score among the samples; (h) CRC cohort:
Histologically confirmed incurable, advanced adenocarcinoma of the
colon or rectum (Tumors of appendiceal origin are not eligible). At
least 5 patients with microsatellite instability-high (MSI-H)
tumors (e.g., as described above) by local laboratory testing may
be enrolled in this cohort; and (i) OC cohort: Histologically
confirmed incurable, advanced epithelial ovarian, fallopian tube,
or primary peritoneal cancer. Borderline ovarian epithelial
neoplasms (e.g., tumors of low malignant potential, atypical
proliferative tumors) are excluded.
[0589] General inclusion criteria include the following:
(a) Age .gtoreq.18 years; (b) Eastern Cooperative Oncology Group
(ECOG) performance status of 0 or 1 (see Table C below for a
description of this 0-5 point scale); (c) Life expectancy
.gtoreq.12 weeks; (d) Adequate hematologic and end organ function,
defined by the following laboratory results obtained within 14 days
prior to the first study treatment (Cycle 1, Day 1): [0590] (i)
Absolute neutrophil count (ANC) .gtoreq.1500 cells/.mu.L; [0591]
(ii) White blood cell (WBC) counts .gtoreq.2,500/.mu.L; [0592]
(iii) Lymphocyte count .gtoreq.500/.mu.L; [0593] (iv) Platelet
count .gtoreq.100,000/.mu.L (without transfusion within 14 days
prior to Cycle 1, Day 1); [0594] (v) Hemoglobin .gtoreq.9.0 g/dL
(patients may be transfused or receive erythropoietic treatment to
meet this criterion); [0595] (vi) Total bilirubin
.ltoreq.1.5.times.upper limit of normal (ULN); [0596] (vii)
Aspartate aminotransferase (AST) and alanine aminotransferase
(ALT).ltoreq.3.0.times.ULN [0597] (viii) Alkaline phosphatase
.ltoreq.2.5.times.ULN with the following exception: Patients with
documented liver or bone metastases: alkaline phosphatase
.ltoreq.5.times.ULN; [0598] (ix) Serum albumin .gtoreq.2.5 g/dL;
[0599] (x) Prothrombin time (PT) and activated partial
thromboplastin time (aPTT).ltoreq.1.5.times.ULN (This applies only
to patients who do not receive therapeutic anticoagulation;
patients receiving therapeutic anticoagulation should be on a
stable dose); [0600] (xi) Measured or calculated creatinine
clearance .gtoreq.50 mL/min on the basis of the Cockcroft-Gault
glomerular filtration rate estimation:
[0600] ( 140 - age ) .times. ( weight in kg ) .times. ( 0.85 if
female ) 72 .times. ( serum creatinine in mg / dL ) ;
##EQU00001##
(e) Serum pregnancy test for women of childbearing potential
(including women who have had a tubal ligation) must be performed
and documented as negative within 14 days prior to Cycle 1, Day 1;
(f) For women who are not postmenopausal (.gtoreq.12 months of
non-therapy-induced amenorrhea) or surgically sterile (absence of
ovaries and/or uterus): agreement to remain abstinent or use single
or combined contraceptive methods that result in a failure rate of
<1% per year during the treatment period and for at least 90
days after the last dose of study drug. Abstinence is only
acceptable if it is in line with the preferred and usual lifestyle
of the patient. Periodic abstinence (e.g., calendar, ovulation,
symptothermal, or postovulation methods) and withdrawal are not
acceptable methods of contraception. Examples of contraceptive
methods with a failure rate of <1% per year include tubal
ligation, male sterilization, hormonal implants, established,
proper use of combined oral or injected hormonal contraceptives,
and certain intrauterine devices. Alternatively, two methods (e.g.,
two barrier methods such as a condom and a cervical cap) may be
combined to achieve a failure rate of <1% per year. Barrier
methods must always be supplemented with the use of a spermicide.
(g) For men: agreement to remain abstinent or use a condom plus an
additional contraceptive method that together result in a failure
rate of <1% per year during the treatment period and for at
least 90 days after the last dose of study drug and agreement to
refrain from donating sperm during this same period. Men with a
pregnant partner must agree to remain abstinent or use a condom for
the duration of the pregnancy. Abstinence is only acceptable if it
is in line with the preferred and usual lifestyle of the patient.
Periodic abstinence (e.g., calendar, ovulation, symptothermal, or
postovulation methods) and withdrawal are not acceptable methods of
contraception.
TABLE-US-00020 TABLE C Eastern Cooperative Oncology Group (ECOG)
performance status scale Grade Description 0 Fully active, able to
carry on all predisease performance without restriction 1
Restricted in physically strenuous activity but ambulatory and able
to carry out work of a light or sedentary nature; e.g., light
housework or office work 2 Ambulatory and capable of all self-care
but unable to carry out any work activities; up and about >50%
of waking hours 3 Capable of only limited self-care, confined to a
bed or chair >50% of waking hours 4 Completely disabled; cannot
carry on any self-care; totally confined to bed or chair 5 Dead
[0601] In addition, patients who meet any of the following
exclusion criteria are excluded from study entry. Types of
exclusion criteria include cancer-specific, treatment-specific, and
general exclusion criteria.
[0602] Cancer-specific exclusion criteria include the
following:
(a) Any anti-cancer therapy, including chemotherapy, hormonal
therapy, or radiotherapy, within 3 weeks prior to initiation of
study treatment, with the following exceptions: [0603] (i) Hormonal
therapy with gonadotropin-releasing hormone (GnRH) agonists or
antagonists for prostate cancer; [0604] (ii) Hormone-replacement
therapy or oral contraceptives; [0605] (iii) Herbal therapy >1
week before Cycle 1, Day 1 (herbal therapy intended as anti-cancer
therapy must be discontinued at least 1 week before Cycle 1, Day
1); [0606] (iv) Palliative radiotherapy for painful metastases or
metastases in potentially sensitive locations (e.g., epidural
space) >2 weeks prior to Cycle 1, Day 1; [0607] (v) Tyrosine
kinase inhibitors (TKIs) approved for treatment of NSCLC that have
been discontinued >7 days prior to Cycle 1, Day 1. Baseline
scans must be obtained after discontinuation of prior TKIs; (b)
Eligibility based on prior treatment with cancer immunotherapy
(CIT) depends on the mechanistic class of the drug and the cohort
for which the patient is being considered, as described below:
[0608] (i) Dose-escalation cohorts: Prior treatment with
immunomodulatory monoclonal antibodies (mAbs) or mAb-derived
therapies is allowed provided that no immune-related Grade
.gtoreq.3 adverse events (other than Grade 3 endocrinopathy managed
with replacement therapy) were observed and at least 5 elimination
half-lives of the drug have elapsed between the last dose of prior
treatment and the proposed Cycle 1, Day 1; [0609] (ii) Expansion
cohorts other than Part I and Part II biopsy cohorts: Prior
treatment with immunomodulatory monoclonal antibodies (mAbs) or
mAb-derived therapies is allowed provided that no immune-related
Grade .gtoreq.3 adverse events (other than Grade 3 endocrinopathy
managed with replacement therapy) were observed and at least 6
weeks have elapsed between the last dose of prior treatment and the
proposed Cycle 1, Day 1, with the following exceptions: prior OX40
agonists are not allowed, and prior PD-L1/PD-1 pathway inhibitors
are not allowed; [0610] (iii) All cohorts: Prior treatment with
cancer vaccines, cytokines, toll-like receptor (TLR) agonists, and
inhibitors of indoleamine 2,3-dioxygenase or
tryptophan-2,3-dioxygenase (IDO/TDO) is allowed provided that no
immune-related Grade .gtoreq.3 adverse events (other than Grade 3
endocrinopathy managed with replacement therapy) were observed and
at least 6 weeks or 5 half-lives of the drug, whichever is shorter,
have elapsed between the last dose and the proposed Cycle 1, Day 1.
Minimum washout is 3 weeks for any prior systemic cancer therapy.
CIT not explicitly described in this protocol should be discussed
with the Medical Monitor to determine potential eligibility; [0611]
(iv) Serial biopsy cohorts: Prior treatment with immunomodulatory
monoclonal antibodies (mAbs) or mAb-derived therapies is allowed
provided that no immune-related Grade .gtoreq.3 adverse events
(other than Grade 3 endocrinopathy managed with replacement
therapy) were observed and at least 6 weeks have elapsed between
the last dose of prior treatment and the proposed Cycle 1, Day 1.
The treatment history of patients enrolling in the Expansion Part I
and Expansion Part II serial biopsy cohorts may be monitored such
that approximately half of the patients in each cohort are naive to
prior OX40 and PD-L1/PD-1 pathway agents; (c) Adverse events from
prior anti-cancer therapy that have not resolved to Grade .ltoreq.1
except for alopecia or endocrinopathy managed with replacement
therapy. Any Grade .ltoreq.2 immune-related adverse event related
to prior immunomodulatory therapy must have resolved completely.
Patients treated with corticosteroids for immune-related adverse
events must demonstrate absence of related symptoms or signs for
.gtoreq.4 weeks following discontinuation of corticosteroids; (d)
Primary central nervous system (CNS) malignancy, or
untreated/active CNS metastases (progressing or requiring
anticonvulsants or corticosteroids for symptomatic control): [0612]
(i) Patients with a history of treated CNS metastases are eligible,
provided they meet all of the following criteria: measurable
disease outside the CNS; radiographic demonstration of improvement
upon the completion of CNS-directed therapy and no evidence of
interim progression between the completion of CNS-directed therapy
and the screening radiographic study; the screening CNS
radiographic study is .gtoreq.4 weeks since completion of
radiotherapy; corticosteroids and anticonvulsants discontinued for
.gtoreq.2 weeks prior to enrollment with no ongoing symptoms
attributable to CNS metastases (anticonvulsants at a stable dose
are allowed); (e) Leptomeningeal disease; (f) Uncontrolled
tumor-related pain: [0613] (i) Symptomatic lesions amenable to
palliative radiotherapy (e.g., bone metastases or metastases
causing nerve impingement) should be treated prior to enrollment;
and [0614] (ii) Asymptomatic metastatic lesions whose further
growth would likely cause functional deficits or intractable pain
(e.g., epidural metastasis that is not currently associated with
spinal cord compression) should be considered for loco-regional
therapy if appropriate prior to enrollment; (g) Uncontrolled
pleural effusion, pericardial effusion, or ascites requiring
recurrent drainage procedures (once monthly or more frequently)
(Patients with indwelling catheters, e.g., PleurX, are allowed);
(h) Malignancies other than disease under study within 5 years
prior to Cycle 1, Day 1, with the exception of those with a
negligible risk of metastasis or death (such as adequately treated
carcinoma in situ of the cervix, basal or squamous cell skin
cancer, localized prostate cancer, or ductal carcinoma in
situ).
[0615] Treatment-specific exclusion criteria include the
following:
(a) History of autoimmune disease, including but not limited to
systemic lupus erythematosus, rheumatoid arthritis, inflammatory
bowel disease, vascular thrombosis associated with antiphospholipid
syndrome, Wegener's granulomatosis, Sjogren's syndrome, Bell's
palsy, Guillain-Barre syndrome, multiple sclerosis, vasculitis, or
glomerulonephritis, with the following caveats: [0616] (i) Patients
with a history of autoimmune hypothyroidism on a stable dose of
thyroid replacement hormone may be eligible; [0617] (ii) Patients
with stable vitiligo may be eligible; (b) Treatment with systemic
immunosuppressive medications (including but not limited to
prednisone, cyclophosphamide, azathioprine, methotrexate,
thalidomide, and TNF.alpha. antagonists) within 2 weeks prior to
Cycle 1, Day 1. (c) Patients who have received acute, low-dose,
systemic immunosuppressant medications (e.g., a one-time dose of
dexamethasone for nausea) may be enrolled in the study after
discussion with and approval by the Medical Monitor: [0618] (i) The
use of inhaled corticosteroids is allowed; [0619] (ii) The use of
mineralocorticoids (e.g., fludrocortisone) for patients with
orthostatic hypotension is allowed; and [0620] (iii) Physiologic
doses of corticosteroids for adrenal insufficiency are allowed; (d)
History of idiopathic pulmonary fibrosis, pneumonitis (including
drug induced), organizing pneumonia (i.e., bronchiolitis
obliterans, cryptogenic organizing pneumonia, etc.), or evidence of
active pneumonitis on screening chest CT scan (History of radiation
pneumonitis in the radiation field (fibrosis) is permitted); (e)
Positive test for HIV infection; (f) Active hepatitis B (defined as
having a positive hepatitis B surface antigen [HBsAg] test at
screening). Patients with past or resolved hepatitis B infection
(defined as having a negative HBsAg test and a positive IgG
antibody to hepatitis B core antigen [anti-HBc]) are eligible; (g)
Active hepatitis C (Patients positive for hepatitis C virus (HCV)
antibody are eligible only if PCR is negative for HCV RNA); (h)
Active tuberculosis; (i) Severe infections within 4 weeks prior to
Cycle 1, Day 1, including but not limited to hospitalization for
complications of infection, bacteremia, or severe pneumonia; (j)
Signs or symptoms of infection within 2 weeks prior to Cycle 1, Day
1; (k) Received oral or IV antibiotics within 2 weeks prior to
Cycle 1, Day 1. Patients receiving prophylactic antibiotics (e.g.,
for prevention of a urinary tract infection or chronic obstructive
pulmonary disease) are eligible; (l) Prior allogeneic bone marrow
transplantation or prior solid organ transplantation; (m)
Administration of a live, attenuated vaccine within 4 weeks before
Cycle 1, Day 1 or anticipation that such a live attenuated vaccine
may be required during the study. Influenza vaccination should be
given during influenza season only. Patients must not receive live,
attenuated influenza vaccine (e.g., FluMist.RTM.) within 4 weeks
prior to Cycle 1, Day 1 or at any time during the study; (n)
History of severe allergic, anaphylactic, or other hypersensitivity
reactions to chimeric or humanized antibodies or fusion
proteins.
[0621] General exclusion criteria include the following:
(a) Inability to comply with study and follow-up procedures; (b)
Pregnancy, lactation, or breastfeeding. Serum pregnancy test (for
women of childbearing potential, including women who have had a
tubal ligation) must be performed and documented as negative within
14 days prior to Cycle 1, Day 1; (c) Significant cardiovascular
disease, such as New York Heart Association cardiac disease (Class
II or greater), myocardial infarction within the previous 3 months,
unstable arrhythmias, or unstable angina; (d) Known clinically
significant liver disease, including active viral, alcoholic, or
other hepatitis, cirrhosis, and inherited liver disease; (e) Major
surgical procedure within 28 days prior to Cycle 1, Day 1 or
anticipation of need for a major surgical procedure during the
course of the study; (f) Any other diseases, metabolic dysfunction,
physical examination finding, or clinical laboratory finding giving
reasonable suspicion of a disease or condition that contraindicates
the use of an investigational drug or that may affect the
interpretation of the results or render the patient at high risk
from treatment complications.
Dose Escalation Stage
[0622] As set forth above and illustrated in FIG. 1, patients are
enrolled in a dose-escalation stage and an expansion stage.
[0623] Approximately 18 to 30 patients may be enrolled in the
dose-escalation stage. Cohorts of at least 3 patients each may be
treated at escalating doses of MOXR0916 in combination with a fixed
dose of MPDL3280A (1200 mg) in accordance with the dose-escalation
rules described below to determine the MTD or maximum administered
dose (MAD). Enrollment of the first two patients in each
dose-escalation cohort may be staggered such that their respective
Cycle 1, Day 1 treatments are administered .gtoreq.72 hours
apart.
[0624] Initially, the dose-limiting toxicity (DLT) assessment
window is 21 days (Days 1-21 of Cycle 1). If a delayed DLT is
observed (e.g., as described herein), the DLT assessment window may
be extended to 42 days after the first administration of MOXR0916
and MPDL3280A for all patients in that cohort and any subsequent
dose-escalation cohorts. Adverse events identified as DLTs or
delayed DLTs are reported to the Sponsor within 24 hours.
[0625] Any dose-escalation stage patient who does not complete the
DLT assessment window (either 21 or 42 days, depending on the DLT
assessment window in effect at the time) for a reason other than a
DLT is considered non-evaluable for dose-escalation decisions and
the MTD assessment and may be replaced by an additional patient at
that same dose level. Patients who receive supportive care during
the DLT assessment window that confounds the evaluation of DLTs
(not including supportive care described below as part of the DLT
definition) may be replaced at the discretion of the Medical
Monitor. A patient who has any component of study treatment held
during the DLT assessment window for a reason other than a DLT such
that administration of the next planned dose is delayed by more
than 7 days, may be considered non-evaluable for dose-escalation
decisions and the MTD assessment and may be replaced by an
additional patient at that same dose level.
[0626] Any one of the following adverse events is considered a DLT
if it occurs during the DLT assessment window in a patient enrolled
in a dose-escalation cohort and is assessed by the investigator to
be related to study treatment:
(a) Grade .gtoreq.3 non-hematologic, non-hepatic adverse event,
with the following exceptions: [0627] (i) Grade 3 nausea, vomiting,
or diarrhea that resolves to Grade .ltoreq.2 with standard-of-care
therapy in .ltoreq.3 days; [0628] (ii) Grade 3 fatigue that
resolves to Grade .ltoreq.2 in .ltoreq.3 days; [0629] (iii) Grade 3
fever (>40 degrees C. for .ltoreq.24 hours); [0630] (iv) Grade 3
adverse event of tumor flare (defined as local pain, irritation, or
rash localized at sites of known or suspected tumor) that resolves
to Grade .ltoreq.2 in .ltoreq.7 days; [0631] (v) Grade 3 laboratory
abnormalities that are asymptomatic and considered by the
investigator not to be clinically significant that resolve to Grade
.ltoreq.2 in .ltoreq.7 days; [0632] (vi) Grade 3 rash that resolves
to Grade .ltoreq.2 in .ltoreq.7 days with therapy equivalent to
prednisone 10 mg/day or less; (b) Grade .gtoreq.4 neutropenia
(absolute neutrophil count [ANC]<500/.mu.L) lasting >7 days;
(c) Grade .gtoreq.3 febrile neutropenia; (d) Grade .gtoreq.4
anemia; (e) Grade .gtoreq.4 thrombocytopenia, or Grade 3
thrombocytopenia associated with clinically significant bleeding;
(f) Grade .gtoreq.3 elevation of serum hepatic transaminase
(alanine aminotransferase [ALT] or aspartate aminotransferase
[AST]) lasting >7 days. For patients with Grade 1 ALT or AST
elevation at baseline as a result of liver metastases, only a Grade
.gtoreq.3 elevation that is also .gtoreq.3.times. baseline lasting
>7 days may be considered a DLT; (g) Grade .gtoreq.3 elevation
of serum bilirubin; and (h) ALT or AST >3.times.upper limit of
normal (ULN) AND total bilirubin >2.times.ULN.
[0633] A delayed DLT is defined as an adverse event that meets one
of the above DLT criteria but occurs between 3 and 6 weeks after
the first administration of MOXR0916 and MPDL3280A (Study Days
22-42).
[0634] The starting dose of MOXR0916 is 0.8 mg, administered by IV
infusion every 21 days to patients in the first cohort. The
escalation increment between successive dose levels is no greater
than 4-fold between successive dose levels, and the proposed doses
for evaluation are 0.8 mg, 3.2 mg, 12 mg, 40 mg, 130 mg, 400 mg,
and 1200 mg. Depending on new nonclinical efficacy, clinical
safety, and PK data, intermediate dose levels of MOXR0916 may be
evaluated. MPDL3280A is administered at a fixed dose of 1200 mg IV
every 21 days.
[0635] In addition to any DLTs, other available relevant
demographic, adverse event, laboratory, dose administration, and
PK/PD data are reviewed prior to all dose-escalation decisions,
which are made by the Medical Monitor in consultation with the
Principal Investigators and a committee composed of the following
Sponsor representatives: safety scientist, statistician, and PK
scientist. Based on review of these emergent clinical data,
intermediate dose levels may be evaluated.
[0636] Dose escalation occurs in accordance with the rules listed
below irrespective of the duration of the DLT window:
(a) A minimum of 3 patients is initially enrolled in each cohort;
(b) If none of the first 3 DLT-evaluable patients experiences a
DLT, enrollment of the next cohort at the next highest dose level
may proceed; (c) If 1 of the first 3 DLT-evaluable patients
experiences a DLT, the cohort is expanded to 6 patients. If there
are no further DLTs in the first 6 DLT-evaluable patients,
enrollment of the next cohort at the next highest dose level may
proceed; (d) If 2 or more of the first 6 DLT-evaluable patients in
a cohort experience a DLT, the MTD is exceeded and dose escalation
stops. An additional 3 patients are then evaluated for DLTs at the
preceding dose level, unless 6 patients have already been evaluated
at that level. However, if the dose level at which the MTD is
exceeded is .gtoreq.2-fold higher than the preceding dose level, 6
patients may be evaluated at an intermediate dose level; (e) If the
MTD is exceeded at any dose level, the highest dose at which fewer
than 2 of 6 DLT-evaluable patients (i.e., <33%) experience a DLT
is declared the MTD; (f) If the MTD is not exceeded at any dose
level, the highest dose administered in this study is declared the
MAD; (g) Any dose level may be expanded beyond 3 patients in the
absence of a DLT if warranted based on Sponsor and investigator
evaluation of non-DLT adverse events, including events occurring
after Cycle 1 and events observed in the expansion cohorts; and (h)
If two or more patients in a single cohort experience Grade
.gtoreq.2 adverse events attributed to study treatment or one or
more AEs meeting the criteria for DLT are observed at any time
during study treatment, the dose may be increased by no more than
2-fold between dose levels for any subsequent dose escalation.
[0637] In addition, the following rules apply specifically to the
first instance in which a delayed DLT is observed. The dose level
at which the delayed DLT was observed is referred to as the "index"
dose level or cohort:
(a) Enrollment at or above the index dose level is temporarily
suspended, unless the index cohort has enrolled fewer than 3
patients, in which case a total of 3 patients may be initially
enrolled in that cohort; (b) The DLT assessment window is extended
to 42 days after the first administration of study treatment. This
extended window is effective immediately for patients already
enrolled at or above the index dose level. Any subsequent
enrollment and dose escalation may proceed according to the general
rules above, with a 42-day assessment window; and (c) Patients who
have been enrolled at a dose level higher than the index dose level
have the option to reduce their dose to a lower dose level, at the
discretion of the investigator. A patient who undergoes dose
reduction prior to completing the DLT assessment window and does
not experience a DLT may be considered non-evaluable for
dose-escalation decisions and the MTD assessment. If a DLT occurs
after such dose reduction may within 42 days of the initial
treatment at the dose level higher than the index dose level, the
DLT may be assigned to the originally assigned dose level.
[0638] Based on available preliminary safety and PK data (collected
in this study or in the ongoing Phase Ia Study G029313 of
single-agent MOXR0916), dose escalation may be halted or modified
by the Sponsor as deemed appropriate. The MOXR0916 dose
administered in this study may not exceed either the highest dose
administered or the MTD of Study G029313.
Expansion Stage
[0639] Approximately 166-330 patients are enrolled in the expansion
stage, which includes two parts (FIG. 1).
[0640] Part I includes a cohort of 6-30 patients who are eligible
for serial biopsies (core needle, punch, forceps, or
excisional/incisional). The objectives of Part I are to explore
tumor biomarkers of pharmacodynamic (PD) activity and obtain
additional safety, tolerability, and PK data at multiple dose
levels. The initial MOXR0916 dose level in this cohort may be 3.2
mg or higher (in combination with MPDL3280A 1200 mg) based on
pharmacodynamic biomarker data collected in this study and the
ongoing Study G029313. Enrollment in Part I at the selected initial
dose level can only begin after the escalation cohort treated at
that dose has satisfied the rules permitting further escalation.
Thereafter, enrollment may proceed at or below the highest dose
level that has already cleared its DLT assessment in the
dose-escalation stage.
[0641] Part II includes multiple cohorts to better characterize the
safety, tolerability, PK variability, biomarkers of anti-tumor
activity, and preliminary efficacy of MOXR0916 in combination with
MPDL3280A in specific cancer types. Enrollment in Part II expansion
cohorts may be initiated at a selected dose level at or below the
MAD or MTD of MOXR0916 in combination with MPDL3280A, as determined
by the Sponsor in consultation with study investigators, based on
assessment of accumulating safety, tolerability, clinical PK,
pharmacodynamic, and anti-tumor activity data. Some of these
cohorts require prospective determination of tumor PD-L1 status. As
shown in FIG. 4A, the planned expansion cohorts in Part II may
include approximately:
(a) 20-40 patients with melanoma; (b) 20-40 patients with renal
cell cancer (RCC); (c) 20-40 patients with triple negative breast
cancer (TNBC) (Expansion beyond approximately 20 patients may be
limited to PD-L1-selected patients, based on prospective testing of
tumor tissue during screening or pre-screening); (d) 20-40 patients
with non-small cell lung cancer (NSCLC) (Expansion beyond
approximately 20 patients may be limited to PD-L1-selected
patients, based on prospective testing of tumor tissue during
screening or pre-screening); (e) 20-40 patients with urothelial
bladder cancer (UBC) (Expansion beyond approximately 20 patients
may be limited to PD-L1-selected patients, based on prospective
testing of tumor tissue during screening or pre-screening); (f)
20-40 patients with colorectal cancer (CRC) (At least 5 of these
patients have tumors that are known to be microsatellite
instability-high (MSI-H) by local testing); (g) 20-40 patients with
ovarian cancer (OC); (h) 10-20 patients with tumors amenable to
serial excisional, incisional or punch biopsies; and (i) Up to 40
patients with tumor types without designated cohorts, selected by
the Sponsor in consultation with investigators, may be included in
an additional exploratory "basket" cohort. A maximum of
approximately 10 patients with a particular histology are enrolled
in this cohort unless anti-tumor activity and/or clinical benefit
per investigator is observed.
[0642] In the United States, the Sponsor may provide to the Center
for Devices and Radiological Health (CDRH) performance
characteristics of the assay prior to testing of tumor tissue for
determination of PD-L1 status. In the event that the Expansion Part
I and Expansion Part II biopsy cohorts are available concurrently,
and a patient meets criteria for both cohorts, the patient may be
enrolled in Part II.
[0643] Whereas Part II (with the exception of serial biopsy cohort
dedicated to patients with primary or acquired resistance to
PD-L1/PD-1 blockade) can exclude patients with prior PD-L1/PD-1
inhibitors, Part III is dedicated to patients with solid tumors
whose most recent anti-cancer therapy included PD-L1/PD-1 blockade.
As shown in FIG. 4A, this group of cohorts includes a total of
approximately 60-160 patients with one of the following
malignancies:
(a) Melanoma;
(b) RCC;
(c) NSCLC;
(d) UBC;
(e) TNBC;
[0644] (f) gastric or gastroesophageal junction adenocarcinoma
(GC); (g) head and neck squamous cell carcinoma (HNSCC); and (h)
additional tumor types without designated cohorts, selected by the
Sponsor in consultation with investigators, may be included in an
exploratory "basket" cohort if activity in one or more of the above
diseases is judged to be promising.
[0645] The Sponsor, in consultation with the investigators,
evaluates all available safety data on an ongoing basis to assess
the tolerability of the dose levels studied. If the frequency of
Grade 3 or 4 toxicities observed in an expansion-stage cohort
(including delayed adverse events and events that would otherwise
meet the criteria for a DLT) or other unacceptable toxicities,
suggest that the MTD has been exceeded at that dose level, accrual
at that dose level may be halted in the expansion and escalation
cohorts and, if applicable, further dose escalation may be halted.
Consideration is then given to resuming enrollment in the expansion
stage at a lower dose level. In addition, if accumulating
tolerability, PK, or PD data suggest that the dose level in an
expansion stage cohort is suboptimal for evaluation of anti-tumor
activity, consideration may be given to enrolling new patients in
that cohort to a different dose level. At no time may a dose level
studied in the expansion stage exceed the highest dose level that
has met escalation criteria in the dose-escalation stage.
[0646] Patients enrolled in either of the dedicated expansion-stage
biopsy cohorts may be required to undergo serial tumor biopsies: at
baseline after eligibility criteria (other than the requirement for
available archival tissue) have been fulfilled, and approximately 2
weeks after the first administration of MOXR0916 and MPDL3280A (on
or between Days 15-21 of Cycle 1). Additional biopsies may be
collected at the investigator's discretion, preferably at the time
of radiographic response or progression. In the Expansion Part I
biopsy cohort, tissue biopsy methods may include core needle,
punch, forceps, or excisional/incisional biopsies. In the Expansion
Part II biopsy cohort, punch or excisional/incisional biopsies are
required.
[0647] In either biopsy cohort, a recent archival specimen may be
used in place of a fresh baseline biopsy under the following
circumstances:
(a) The specimen meets the sample criteria (e.g., number of cores
or size of punch); (b) The specimen was collected within 3 months
of the proposed Cycle 1, Day 1; (c) The specimen was collected
subsequent to any systemic therapy or radiation therapy
administered to the relevant anatomic region; (d) The specimen
originates from the same lesion or organ as the proposed site of
the on-treatment biopsy.
[0648] Patients whose baseline biopsy is found to be unevaluable
(i.e., due to insufficient material or lack of tumor cells in the
sample) may decline to undergo an on-treatment biopsy but may
receive study treatment. Such patients, as well as patients whose
on-treatment biopsy is found to be unevaluable, may be replaced for
the purpose of serial biopsy assessment.
[0649] Patients who are enrolled in cohorts other than the
dedicated biopsy cohorts may be asked to undergo optional biopsies
(core needle, punch, forceps, or excisional/incisional) to explore
PD changes related to the activity of MOXR0916 and MPDL3280A. The
recommended biopsy timepoints are the same as described above.
On-treatment biopsies may not be pursued if the baseline sample is
unevaluable and no recent archival specimen is available for
comparison.
Dose Reduction
[0650] There is no dose reduction of MPDL3280A, which is to be
administered at a fixed dose of 1200 mg every 21 days. In general,
there may be no intrapatient dose escalation or dose reduction for
MOXR0916 in this study. However, if available cumulative safety
data suggest that a dose level initially selected for expansion in
combination with MPDL3280A exceeds the MTD, accrual at that dose
level may be halted in the expansion and escalation cohorts and, if
applicable, further dose escalation may be halted. In this
circumstance, individual patients may have the option of dose
reduction to the new dose level of MOXR0916 selected for expansion
in combination with MPDL3280A if the following criteria are met:
The patient's initially assigned dose is equal to (or greater than)
the dose level that has been closed to further enrollment; and the
overall benefit/risk balance favors continued treatment, in the
opinion of the investigator.
Treatment after Disease Progression
[0651] Patients may continue study treatment after standard RECIST
v1.1 criteria for progressive disease are met provided they meet
all the following criteria: absence of symptoms and signs
(including worsening of laboratory values, e.g., new or worsening
hypercalcemia) indicating unequivocal progression of disease; no
decline in ECOG performance status; and absence of tumor
progression at critical anatomical sites that cannot be readily
managed and stabilized by protocol-allowed medical interventions
prior to repeat dosing. Patients provide written consent to
acknowledge discussion with the treating investigator of the
benefit-risk balance of continuing study treatment beyond
radiographic progression.
[0652] If radiographic disease progression is confirmed at a
subsequent tumor assessment, patients may be considered for
continued study treatment at the investigator's discretion after
discussion with the Medical Monitor, if they continue to meet the
criteria above and have evidence of clinical benefit, as evidenced
by at least one of the following: tumor shrinkage (at least 30%
decrease in diameter from baseline) of one or more evaluable
lesions; or improvement in one or more symptoms or signs
attributable to the underlying cancer (e.g., decreased requirement
for narcotics for pain, decreased dyspnea associated with pleural
effusion, weight gain) as assessed by the investigator.
Dosage, Administration, and Compliance
[0653] The approximate dose levels of MOXR0916 proposed to be
evaluated in this study include 0.8, 3.2, 12, 40, 130, 400, and
1200 mg administered every 3 weeks by IV infusion. Intermediate
dose levels of MOXR0916 may be evaluated based on new nonclinical
efficacy, clinical safety, and clinical PK data after consultation
with participating investigators. Doses are not dependent on body
weight.
[0654] The initial dose of MOXR0916 may be delivered over 90.+-.10
minutes (although the infusion may be slowed or interrupted for
patients who experience infusion-associated symptoms), followed by
a 90-minute observation period. If the 90-minute infusion is
tolerated without infusion-associated adverse events, the second
infusion may be delivered over 60.+-.10 minutes, followed by a 60
minute observation period. If the 60-minute infusion is well
tolerated, all subsequent infusions may be delivered over 30.+-.10
minutes, followed by a 30-minute observation period. Patients who
have previously received MOXR0916 on Study G029313 may receive the
initial dose at the fastest rate that was previously tolerated.
There may be no dose reduction for MOXR0916 in this study except as
specified above under "Dose Reduction."
[0655] The dose of MPDL3280A to be administered in combination with
MOXR0916 in this study is 1200 mg IV every 3 weeks. This dose is
fixed and not dependent on body weight.
[0656] MPDL3280A may be administered after the MOXR0916 infusion
and subsequent observation period.
[0657] The initial dose of MPDL3280A may be delivered over 60.+-.10
minutes. If the first infusion is tolerated without
infusion-associated adverse events, the second infusion may be
delivered over 30.+-.10 minutes. If the 30-minute infusion is well
tolerated, all subsequent infusions may be delivered over 30
(.+-.10) minutes. All doses of MPDL3280A may be followed by a
30-minute observation period. Patients who have previously received
MPDL3280A on another clinical trial may receive the initial dose at
the fastest rate that was previously tolerated. There is no dose
reduction for MPDL3280A in this study.
Concomitant Therapy
[0658] Concomitant therapy includes any medication (e.g.,
prescription drugs, over-the-counter drugs, herbal or homeopathic
remedies, nutritional supplements) used by a patient from 7 days
prior to screening to the treatment discontinuation visit (and from
7 days prior to re-screening to the re-treatment discontinuation
visit). All medications should be reported to the investigator and
recorded.
[0659] Patients who experience infusion-associated symptoms may be
treated symptomatically with acetaminophen, ibuprofen,
diphenhydramine, and/or ranitidine or another H2 receptor
antagonist, as per standard practice (for sites outside the U.S.,
equivalent medications may be substituted per local practice).
Serious infusion-associated events manifested by dyspnea,
hypotension, wheezing, bronchospasm, tachycardia, reduced oxygen
saturation, or respiratory distress should be managed with
supportive therapies as clinically indicated (e.g., supplemental
oxygen and .beta.2-adrenergic agonists). Premedication may be
administered for Cycles .gtoreq.2 at the discretion of the treating
physician after consultation with the Medical Monitor.
[0660] Systemic corticosteroids and TNF.alpha. antagonists may
attenuate potential beneficial immunologic effects of treatment
with MOXR0916 and MPDL3280A but may be administered at the
discretion of the treating physician in an emergency or after
consultation with the Medical Monitor. If feasible, alternatives to
corticosteroids should be considered. The use of inhaled
corticosteroids and mineralocorticoids (e.g., fludrocortisone for
patients with orthostatic hypotension or adrenocortical
insufficiency) is allowed. Physiologic doses of corticosteroids for
adrenal insufficiency are allowed. Megestrol administered as an
appetite stimulant is also permitted.
[0661] Patients who use oral contraceptives, hormone-replacement
therapy, prophylactic or therapeutic anticoagulation therapy (such
as low molecular weight heparin or warfarin at a stable dose
level), or other maintenance therapy for non-malignant indications
should continue their use. Males and females of reproductive
potential should use highly effective means of contraception.
[0662] Use of the following therapies is prohibited during the
study:
(a) Any concomitant therapy intended for the treatment of cancer,
whether health authority--approved or experimental, including (but
not limited to) the following: chemotherapy, hormonal therapy,
immunotherapy, radiotherapy, investigational agents, or herbal
therapy; [0663] (i) Radiotherapy may be considered for pain
palliation (e.g., treatment of known bony metastases) if patients
are otherwise deriving benefit. For patients in a dose escalation
cohort, palliative radiotherapy should be deferred until completion
of the DLT assessment window.
[0664] Study treatment administration may be suspended during
radiotherapy with agreement from the Medical Monitor; [0665] (ii)
Patients experiencing a mixed response may undergo local therapy
(e.g., surgery, stereotactic radiosurgery, radiotherapy,
radiofrequency ablation) for control of three or fewer lesions upon
approval by the Medical Monitor; [0666] (iii) Patients who undergo
radiotherapy to or resection of a target lesion may subsequently
become unevaluable for response determination according to RECIST
v1.1 or modified RECIST; (b) Immunostimulatory agents, including
but not limited to IFN.alpha., IFN.gamma., or IL2, during the
entire study; (c) Immunosuppressive medications, including but not
limited to cyclophosphamide, azathioprine, methotrexate, and
thalidomide; and (d) Granulocyte colony-stimulating factors (e.g.,
granulocyte colony-stimulating factor, granulocyte macrophage
colony-stimulating factor, and/or pegfilgrastim); (e) traditional
herbal medicines; and (f) receptor activator of nuclear factor
kappa B (RANK) inhibitor (i.e., denosumab). Patients who are
receiving denosumab prior to enrollment must be willing and
eligible to receive a bisphosphonate instead while on study.
Outcome Measures
[0667] The safety and tolerability of MOXR0916 and MPDL3280A is
assessed using the following primary safety outcome measures:
incidence and nature of DLTs; and incidence, nature, and severity
of adverse events graded according to NCI CTCAE v4.0.
[0668] In addition, safety may be assessed using the following
secondary safety outcome measures: incidence of anti-MOXR0916
antibodies and anti-MPDL3280A antibodies and the potential
correlation with PK, PD, and safety parameters; change in vital
signs; change in clinical laboratory results, including ECGs; and
number of cycles received and dose intensity.
[0669] The following pharmacokinetic (PK) parameters may be derived
from concentration-time profile of MOXR0916 and MPDL3280A following
administration, when appropriate as data allow: total exposure
(AUC); Cmax; Cmin; CL; and Vss. Other parameters such as
accumulation ratio, half-life, and dose proportionality may also be
calculated.
[0670] The following activity outcome measures may be assessed:
(a) Objective response, defined as a complete response (CR) or
partial response (PR) confirmed .gtoreq.4 weeks after initial
documentation, determined using RECIST v.1.1; (b) Duration of
objective response, defined as the time from the first occurrence
of a documented, objective response until the time of relapse or
death from any cause, determined using RECIST v.1.1; (c)
Progression-free survival (PFS), defined as the time from the first
study treatment (Day 1) to the first occurrence of progression or
death from any cause, whichever occurs first, determined using
RECIST v.1.1; (d) Objective response, duration of objective
response, and PFS determined using modified RECIST; (e) Overall
survival (OS), defined as the time from first study treatment to
death from any cause.
[0671] The following exploratory PD outcome measures may be
assessed: changes in T, B, and NK cell numbers (T, B, and NK assay)
in blood; changes in prevalence of various immune cell
subpopulations in blood (e.g., effector/memory T cells, regulatory
T cells, and MDSCs); changes in activation, proliferation, and
functional status of T-cell subsets in blood; identification and
profiling of exploratory biomarkers in plasma (i.e., interleukin-2
[IL2], IFN.gamma., and other markers); changes in
tumor-infiltrating CD8+ T cells (and other exploratory markers) in
freshly obtained tumor tissue prior to and during study treatment;
and changes in tumor-infiltrating T-cell activity (measured by
expression of granzyme B and other markers) in freshly obtained
tumor tissue prior to and during study treatment.
[0672] The following additional exploratory biomarker outcome
measures may be assessed when appropriate: status of PD-L1 and OX40
(and other exploratory markers) in tumor tissue; status of immune
infiltrate in tumor tissue, including enumeration and
characterization of various immune cell subpopulations; and
analysis of single nucleotide polymorphisms (SNPs) in genes
including but not limited to those that encode Fc receptors.
Study Assessments
[0673] A complete physical examination performed at screening
should include an evaluation of the head, eyes, ears, nose, and
throat, and the cardiovascular, dermatological, musculoskeletal,
respiratory, gastrointestinal, genitourinary, and neurological
systems. Any abnormality identified at baseline should be
recorded.
[0674] At subsequent visits (or as clinically indicated), limited,
symptom-directed physical examinations should be performed. Changes
from baseline abnormalities should be recorded in the patient's
medical record. New or worsened clinically significant
abnormalities should be recorded as adverse events.
[0675] As part of tumor assessments, the physical exam should also
include evaluation for lymphadenopathy, splenomegaly, hepatomegaly,
and cutaneous neoplasms or metastases. All patients should be
monitored for symptoms of CNS metastases and such reported symptoms
should be followed by a full neurological examination. A brain MRI
or contrast enhanced head CT should be done as clinically indicated
to confirm or refute new or worsening brain involvement.
[0676] All known sites of disease must be documented at screening
and re-assessed at each subsequent tumor evaluation. Screening and
subsequent tumor assessments must include CT scans (with IV
contrast unless contraindicated and oral contrast as appropriate
per institutional standards) or MRI of the chest, abdomen, and
pelvis. If a CT scan for tumor assessment is performed in a
positron emission tomography (PET)/CT scanner, the CT acquisition
must be consistent with the standards for a full-contrast CT scan.
Brain imaging (either MRI or contrast-enhanced CT) is required at
screening for patients with treated brain metastases and as
clinically indicated based on symptoms or signs suggestive of new
or worsening CNS metastases. In the event of an equivocal head CT,
a brain MRI is required to clarify the presence or extent of
suspected brain metastases. Further investigations such as bone
scans and CT scans of the neck should also be performed if there is
any clinical suspicion of disease at any site that may not be
demonstrated by the minimum schedule of assessments listed above.
At the investigator's discretion, other methods of assessment of
measurable disease as per RECIST v1.1 may be used.
[0677] The same radiographic procedures used to assess disease
sites at screening should be used throughout the study (e.g., the
same contrast protocol for CT scans). Response may be assessed by
the investigator on the basis of physical examinations and the
imaging modalities detailed above, using both RECIST v1.1 and
modified RECIST criteria. Assessments should be performed by the
same evaluator if possible to ensure internal consistency across
visits.
[0678] Patients who continue treatment beyond radiographic disease
progression per RECIST v1.1 may be monitored with a follow-up scan
in 6 (.+-.2) weeks (i.e., at the next scheduled tumor assessment
when the scan frequency is every 2 cycles or as an unscheduled
tumor assessment when the scan frequency is every 4 cycles), or
earlier if clinically indicated. Tumor assessments should be
continued every 2 cycles thereafter until two consecutive scans
demonstrate stability or improvement with respect to the first scan
that showed radiographic disease progression, at which point the
scan frequency should revert or transition to every 4 cycles if
applicable.
[0679] After initial study treatment discontinuation, follow-up
tumor assessments may be performed until death, disease
progression, initiation of another systemic anti-cancer therapy,
loss to follow-up, withdrawal of consent, or study termination,
whichever occurs first.
[0680] FDG-PET/CT imaging scans may be acquired at baseline and at
the time of the first tumor assessment. In addition, an optional
FDG-PET/CT scan may be performed at the first evidence of
radiographic disease progression to assess whether apparent
increases in tumor volume related to immunomodulatory activity of
MOXR0916 and MPDL3280A (i.e., pseudoprogression) may be
distinguished from neoplastic proliferation and disease
progression. PET/CT scans at other timepoints are optional. All
FDG-PET/CT scans are to be acquired in accordance with the
specification provided in the imaging manual. A combination PET and
CT scanner should be used for all acquisitions. The baseline
FDG-PET/CT scan should be performed during the screening period
only after all other inclusion and exclusion criteria have been
satisfied, unless it was integrated with a diagnostic quality
full-contrast CT scan that fulfills the screening tumor assessment
requirement. All FDG-PET/CT scans should be acquired before any
scheduled invasive procedure such as a tumor biopsy if at all
possible (biopsy location may need to be noted to ensure accurate
assessment during central PET imaging review).
[0681] The planned duration of the study is approximately 3 years.
The end of this study is defined as the completion of the 90-day
adverse event reporting period for the last patient receiving study
treatment. This is expected to occur approximately 12 months after
the last patient has been enrolled.
Example 2
A Phase Ib Dose Escalation Study of the OX40 Agonist MOXR0916 and
the PD-L1 Inhibitor Atezolizumab in Patients with Advanced Solid
Tumors
Background
[0682] OX40 is a co-stimulatory receptor that is transiently
expressed by T cells upon antigen recognition. In murine models,
OX40 engagement by an agonist anti-OX40 antibody can promote
durable tumor regression associated with co-stimulation of effector
T cells and reduction of regulatory T cells. MOXR0916 is a
humanized effector-competent agonist IgG1 monoclonal antibody (mAb)
that targets OX40, and atezolizumab is an engineered humanized IgG1
mAb that targets PD-L1. The purpose of this study is to examine the
safety and pharmacokinetics (PK) of agonist anti-OX40 antibody
treatment in combination with anti-PD-L1 antibody treatment.
Methods
[0683] A Phase I, open-label, multicenter study was conducted as
described in Example 1 to evaluate the safety and PK of MOXR0916
and atezolizumab in patients (pts) with locally advanced or
metastatic solid tumors (FIG. 4A). A 3+3 dose-escalation was
conducted with a 21-day window to evaluate dose-limiting toxicity
(DLT). Escalating doses of MOXR0916 in combination with a fixed
1200 mg dose of atezolizumab were administered every 3 weeks (q3w).
An expansion cohort to enable immune profiling of serial tumor
biopsies was also enrolled. Prior immunotherapy with adequate
washout was allowed if there was no history of Grade (G) .gtoreq.3
immune-mediated adverse events (AEs).
Results
[0684] 25 patients were treated in 7 dose escalation cohorts (dose
levels 0.8 to 600 mg) and 19 additional patients were treated in a
serial biopsy cohort. The median number of prior therapies for
metastatic disease was 2 (range 0-7), and 5 patients had received
prior PD-1/PD-L1 antibodies. No DLTs, G4/5 AEs attributed to study
treatment, or related AEs leading to treatment discontinuation were
reported. The majority of treatment-related AEs were G1 in
severity; 1 related G3 event (pneumonitis responsive to
corticosteroids) was reported. The PK of each mAb was consistent
with its established single agent profile.
[0685] In a parallel phase I single-agent MOXR0916 study, at doses
.gtoreq.40 mg q3w, MOXR0916 PK was linear and consistent with IgG1
mAb (FIG. 2) and sustained peripheral blood OX40 receptor
saturation was achieved (FIGS. 3A-G). Dose-dependent peripheral
receptor occupancy was observed, with continuous peripheral OX40
saturation achieved at doses .gtoreq.40 mg. Doses .gtoreq.200 mg
are projected to achieve continuous tumor OX40 saturation in cycle
1 (95% occupancy at trough assuming 20:1 blood:tumor partioning).
PD-L1 expression increased post MOXR0916 treatment in RCC, NSCLC,
melanoma, and cervical tumors.
[0686] A transient, bimodal increase in plasma cytokines was
observed with MOXR0916+Atezolizumab treatment. The observed early
(6 hr, C1D2) increase in IP-10 and comparatively lower increase in
IFN.gamma. may be attributable to MOXR0916. The observed increase
in IP-10 and comparatively higher increase in IFN.gamma. at C1D15
may be attributed to atezolizumab or MOXR+atezolizumab.
[0687] Objective responses were observed, including two PRs in
PD(L)-1-naive patients diagnosed with RCC and bladder cancer,
respectively. The RCC patient was observed with a confirmed partial
response (PR) after commencement in the MOXR0916 300
mg+atezolizumab 1200 mg q3w study described above. The patient had
previously been part of a phase I single-agent MOXR0916 study for 8
cycles (i.e., approximately 24 weeks) with a best response of
stable disease. The bladder cancer patient was part of the first
dose escalation cohort (see FIG. 4A). The regimen selected for dose
expansion was MOXR0916 300 mg+atezolizumab 1200 mg q3w.
[0688] 10% of all patients enrolled in the MOXR0916 and
atezolizumab study had received prior treatment with an OX40
agonist (e.g., an anti-OX40 agonist antibody), and 18% of all
patients enrolled in the MOXR0916 and atezolizumab study had
received prior treatment with a PD-L1 or PD-1 inhibitor (e.g., an
anti-PD-L1 or anti-PD-1 antibody). In the MOXR0916 and atezolizumab
study, evidence of immune activation was observed in patients
previously treated with single-agent OX40 agonist antibody or
single-agent anti-PD-1 antibody in multiple cancer types. For
example, the RCC patient described above, who had previously been
treated with MOXR0916, showed a confirmed partial response with
evidence of pharmacodynamic (PD) modulation in paired tumor
biopsies upon combination treatment with MOXR0916 and atezolizumab
(e.g., PD-L1 status shifting from negative to positive upon
combination treatment). These results demonstrate immune activation
including adaptive upregulation of PD-L1 upon combination treatment
with MOXR0916 and atezolizumab, including PD modulation in patients
whose immediate prior therapy was single-agent OX40 agonist
antibody or single-agent anti-PD-1 antibody.
Conclusions
[0689] The combination of MOXR0916 and atezolizumab was
well-tolerated. An expansion phase, with each agent administered at
its recommended monotherapy dose, is ongoing in selected tumor
types.
Example 3
A Phase Ib, Open Label, Dose Escalation Study of the Safety and
Pharmacokinetics of MOXR0916, Atezolizumab, and Bevacizumab in
Patients with Locally Advanced or Metastatic Solid Tumors
Study Design
[0690] This is a Phase Ib, open label, multicenter, dose escalation
study designed to evaluate the safety, tolerability, and
pharmacokinetics of the combination of MOXR0916, atezolizumab, and
bevacizumab in patients with locally advanced, recurrent, or
metastatic incurable solid malignancy that has progressed after
available standard therapy; or for which standard therapy has
proven to be ineffective or intolerable, or is considered
inappropriate; or for which a clinical trial of an investigational
agent is a recognized standard of care.
[0691] This study consists of a screening period, a treatment
period, and a post treatment follow up period. The study includes a
dose escalation stage and an expansion stage. MOXR0916,
atezolizumab, and bevacizumab are each administered by intravenous
(IV) infusion on Day 1 of 21 day cycles. In the absence of
unacceptable toxicity or clinically compelling disease progression,
treatment with all agents may be continued beyond Cycle 1 based on
a favorable assessment of benefit and risk by the investigator.
[0692] All adverse events are monitored and recorded for at least
90 days after the last dose of study treatment or until initiation
of another systemic anti-cancer therapy, whichever occurs first.
After this period, the Sponsor is notified if the investigator
becomes aware of any serious adverse events if the event is
believed to be related to prior study drug treatment. Adverse
events are graded according to NCI CTCAE v4.0.
[0693] To characterize the PK properties of MOXR0916, atezolizumab,
and bevacizumab and pharmacodynamic responses to treatment, blood
samples are taken at various timepoints before and after dosing.
Patients undergo tumor assessments at screening and during the
study. Patients may be permitted to continue study treatment even
if standard RECIST v1.1 criteria for progressive disease are met,
provided they meet the criteria for continued treatment. All
patients who discontinue study treatment for reasons other than
disease progression (e.g., adverse events) continue tumor
assessments. Patients who discontinue study treatment may return to
the clinic for a treatment discontinuation visit within 30 days
after the last dose of study treatment. All patients are followed
for survival and subsequent anti-cancer therapy information
approximately every 3 months until death, loss to follow up, or
study termination, unless the patient requests to be withdrawn from
follow up.
Study Objectives
[0694] The primary objective for this study is to evaluate the
safety and tolerability of the combination of MOXR0916,
atezolizumab, and bevacizumab, in patients with locally advanced or
metastatic solid tumors.
[0695] The secondary objectives for this study are as follows:
(a) To estimate the MTD of MOXR0916 when administered in
combination with atezolizumab and bevacizumab, and to characterize
the DLTs; (b) To identify a recommended Phase II dose for MOXR0916
administered in combination with atezolizumab and bevacizumab; (c)
To characterize the pharmacokinetics of MOXR0916, atezolizumab, and
bevacizumab when administered in combination; (d) To characterize
the immunogenic potential of MOXR0916, atezolizumab, and
bevacizumab when administered in combination by measuring
anti-MOXR0916, anti-atezolizumab, and anti-bevacizumab antibodies,
respectively, and assessing their relationship with other outcome
measures; and (e) To make a preliminary assessment of the
anti-tumor activity of the combination of MOXR0916, atezolizumab,
and bevacizumab, in patients with locally advanced or metastatic
solid tumors.
[0696] The exploratory objectives for this study are as
follows:
(a) To make a preliminary assessment of biomarkers that might act
as pharmacodynamic indicators of activity of the combination of
MOXR0916, atezolizumab, and bevacizumab, in patients with locally
advanced or metastatic solid tumors; and (b) To make a preliminary
assessment of biomarkers that might act as predictors of anti-tumor
activity of the combination of MOXR0916, atezolizumab, and
bevacizumab, in patients with locally advanced or metastatic solid
tumors.
Study Population
[0697] Cancer specific inclusion criteria include the
following:
(a) Histologic documentation of locally advanced, recurrent or
metastatic incurable solid malignancy that has progressed after
available standard therapy; or for which standard therapy has
proven to be ineffective or intolerable, or is considered
inappropriate; or for which a clinical trial of an investigational
agent is a recognized standard of care; (b) Confirmed availability
of representative tumor specimens in paraffin blocks (preferred) or
.gtoreq.15 unstained slides, with an associated pathology report.
Acceptable samples include core needle biopsies for deep tumor
tissue (minimum three cores) or excisional, incisional, punch, or
forceps biopsies for cutaneous, subcutaneous, or mucosal lesions.
Fine-needle aspiration, brushing, cell pellet from effusions or
ascites, and lavage samples are not acceptable. Tumor tissue from
bone metastases is not evaluable for PD-L1 expression and is
therefore not acceptable. If adequate tissue from distinct time
points (such as time of initial diagnosis and time of disease
recurrence) and/or multiple metastatic tumors is available,
priority is given to the tissue most recently collected (ideally
subsequent to the most recent systemic therapy). Multiple samples
may be collected for a given patient, on the basis of availability;
however, the requirement for a block or .gtoreq.15 unstained slides
is satisfied by a single biopsy or resection specimen. Prior to
signing the main study informed consent form, patients may sign a
pre-screening consent form to specifically allow the collection and
testing of archival or fresh tumor specimens. A patient with
insufficient or unavailable archival tissue may be eligible, upon
discussion with the Medical Monitor, if the patient meets any of
the following: Can provide at least 10 unstained, serial slides; is
willing to consent to and undergo a pretreatment core, punch, or
excisional/incisional biopsy sample collection of the tumor; or is
to be enrolled in a dose-escalation cohort. If the location of the
tumor renders the tumor biopsy medically unsafe, eligibility may be
provided with Medical Monitor approval; (c) Measurable disease per
RECIST v1.1; (d) Dose Escalation Stage: Histologically confirmed
incurable, advanced RCC. Both clear cell and nonclear cell
histologies are permitted in the dose escalation stage. Prior
therapy for RCC, including prior VEGF inhibitors and/or prior
PD-L1/PD-1 inhibitors, are permitted. (e) Dose Expansion Stage: 1L
RCC cohort: Histologically confirmed incurable, advanced RCC with
component of clear cell histology and/or component of sarcomatoid
histology. Patients must not receive prior systemic therapy for
RCC, including adjuvant systemic therapy. Prior treatment with
placebo in the adjuvant setting is allowed. 2L+ RCC cohort:
Histologically confirmed incurable, advanced RCC with component of
clear-cell histology and/or component of sarcomatoid histology.
Patients have demonstrated disease progression during or following
at least one systemic therapy for RCC. Prior VEGF inhibitors and
prior PD-L1/PD-1 inhibitors are permitted.
[0698] Cancer specific exclusion criteria include:
(a) Any anti-cancer therapy, whether investigational or approved,
including chemotherapy, hormonal therapy, or radiotherapy, within 3
weeks prior to initiation of study treatment, with the following
exceptions: [0699] (i) Hormonal therapy with gonadotropin releasing
hormone agonists or antagonists for prostate cancer; [0700] (ii)
Hormone replacement therapy or oral contraceptives; [0701] (iii)
Kinase inhibitors approved as anti-cancer therapy by the FDA or
local health authorities that have been discontinued >7 days
prior to Cycle 1, Day 1; baseline scans are obtained after
discontinuation of prior TKIs and criteria pertaining to adverse
events attributed to prior cancer therapies must be satisfied;
[0702] (iv) Herbal therapy >1 week before Cycle 1, Day 1 (herbal
therapy intended as anti cancer therapy must be discontinued at
least 1 week before Cycle 1, Day 1); and [0703] (v) Palliative
radiotherapy for painful metastases or metastases in potentially
sensitive locations (e.g., epidural space) >2 weeks prior to
Cycle 1, Day 1; (b) Eligibility based on prior treatment with
cancer immunotherapy (CIT) depends on the mechanistic class of the
drug and the cohort for which the patient is being considered, as
described below. In addition, all criteria pertaining to adverse
events attributed to prior cancer therapies must be satisfied:
[0704] MOXR0916+Atezolizumab+Bevacizumab (Triplet) Arm (Dose
Escalation and Expansion Part IV):
Prior treatment with immunomodulatory monoclonal antibodies (mAbs)
or mAb-derived therapies is allowed provided that at least 6 weeks
have elapsed between the last dose of prior treatment and the
proposed Cycle 1 Day 1, with the following exceptions: [0705] (i)
Prior PD-L1/PD-1 pathway inhibitors are not subject to a specific
washout beyond the 3-week minimum for any systemic anti-cancer
therapy; [0706] (ii) Prior OX40 agonists are not subject to a
specific washout beyond the 3-week minimum for any systemic
anti-cancer therapy.
[0707] All Cohorts:
Prior treatment with cancer vaccines, cytokines, toll-like receptor
(TLR) agonists, and inhibitors of indoleamine 2,3-dioxygenase or
tryptophan-2,3-dioxygenase (IDO/TDO) is allowed provided that at
least 6 weeks or 5 half-lives of the drug, whichever is shorter,
have elapsed between the last dose and the proposed Cycle 1, Day 1.
Minimum washout is 3 weeks for any prior systemic cancer therapy.
(c) Any history of an immune related Grade 4 adverse event
attributed to prior cancer immunotherapy (other than endocrinopathy
managed with replacement therapy or asymptomatic elevation of serum
amylase or lipase). Any history of an immune related Grade 3
adverse event attributed to prior cancer immunotherapy (other than
endocrinopathy managed with replacement therapy or asymptomatic
elevation of serum amylase or lipase) that resulted in permanent
discontinuation of the prior immunotherapeutic agent and/or
occurred .ltoreq.6 months prior to Cycle 1 Day 1. Adverse events
from prior anti-cancer therapy that have not resolved to Grade
.ltoreq.1 except for alopecia or endocrinopathy managed with
replacement therapy All immune related adverse events related to
prior immunomodulatory therapy (other than endocrinopathy managed
with replacement therapy or stable vitiligo) must have resolved
completely to baseline. Patients treated with corticosteroids for
immune related adverse events must demonstrate absence of related
symptoms or signs for .gtoreq.4 weeks following discontinuation of
corticosteroids; (d) Primary CNS malignancy, or untreated CNS
metastases or active (progressing or requiring corticosteroids for
symptomatic control) CNS metastases; [0708] (i) Patients with a
history of treated asymptomatic CNS metastases are eligible,
provided they meet all of the following criteria: measurable
disease outside the CNS; no ongoing requirement for corticosteroids
as therapy for CNS disease, with corticosteroids discontinued for
.gtoreq.2 weeks prior to enrollment; anticonvulsants at a stable
dose are allowed; no stereotactic radiation within 7 days or
whole-brain radiation within 14 days prior to Cycle 1, Day 1; no
evidence of interim progression between the completion of CNS
directed therapy and the screening radiographic study. Patients
with new asymptomatic CNS metastases detected at the screening scan
must receive radiation therapy and/or surgery for CNS metastases.
Following treatment, these patients may then be eligible without
the need for an additional brain scan prior to randomization, if
all other criteria are met. (e) Any history of leptomeningeal
disease; (f) Uncontrolled tumor-related pain: [0709] (i)
Symptomatic lesions amenable to palliative radiotherapy (e.g., bone
metastases or metastases causing nerve impingement) are treated
prior to enrollment; and [0710] (ii) Asymptomatic metastatic
lesions whose further growth would likely cause functional deficits
or intractable pain (e.g., epidural metastasis that is not
currently associated with spinal cord compression) are considered
for loco-regional therapy if appropriate prior to enrollment. (g)
Uncontrolled pleural effusion, pericardial effusion, or ascites
requiring recurrent drainage procedures (once monthly or more
frequently). Patients with indwelling catheters (e.g., PleurX) are
allowed; (h) Malignancies other than disease under study within 5
years prior to Cycle 1, Day 1, with the exception of those with a
negligible risk of metastasis or death (such as adequately treated
carcinoma in situ of the cervix, basal or squamous cell skin
cancer, localized prostate cancer, or ductal carcinoma in
situ);
[0711] Treatment-specific exclusion criteria include the
following:
(a) History of autoimmune disease, including but not limited to
systemic lupus erythematosus, rheumatoid arthritis, inflammatory
bowel disease, vascular thrombosis associated with antiphospholipid
syndrome, Wegener's granulomatosis, Sjogren's syndrome, Bell's
palsy, Guillain Barre syndrome, multiple sclerosis, vasculitis, or
glomerulonephritis, with the following caveats: [0712] (i) Patients
with a history of autoimmune hypothyroidism on a stable dose of
thyroid replacement hormone may be eligible; [0713] (ii) Patients
with controlled Type 1 diabetes mellitus on a stable insulin dosing
regimen may be eligible; and [0714] (iii) Patients with eczema,
psoriasis, lichen simplex chronicus, or vitiligo with dermatologic
manifestations only (e.g. no psoriatic arthritis) may be eligible
provided they meet the following conditions: rash must cover less
than 10% of body surface area (BSA); disease is well controlled at
baseline and only requiring low potency topical steroids; no acute
exacerbations of underlying condition within the last 12 months
(not requiring psoralen plus ultraviolet A radiation [PUVA],
methotrexate, retinoids, biologic agents, oral calcineurin
inhibitors, high potency or oral steroids). (b) Treatment with
systemic immunosuppressive medications (including but not limited
to prednisone, cyclophosphamide, azathioprine, methotrexate,
thalidomide, and TNFa antagonists) within 2 weeks prior to Cycle 1,
Day 1; (c) Patients who have received acute, low dose, systemic
immunosuppressant medications (e.g., a one-time dose of
dexamethasone for nausea) may be enrolled in the study after
discussion with and approval by the Medical Monitor; [0715] (i) The
use of inhaled corticosteroids (e.g., fluticasone for chronic
obstructive pulmonary disease) is allowed; [0716] (ii) The use of
oral mineralocorticoids (e.g., fludrocortisone for patients with
orthostatic hypotension or adrenocortical insufficiency) is
allowed; and [0717] (iii) Physiologic doses of corticosteroids for
adrenal insufficiency are allowed. (d) History of idiopathic
pulmonary fibrosis, pneumonitis (including drug induced),
organizing pneumonia (i.e., bronchiolitis obliterans, cryptogenic
organizing pneumonia, etc.), or evidence of active pneumonitis on
screening chest CT scan. History of radiation pneumonitis in the
radiation field (fibrosis) is permitted. History of drug-induced
pneumonitis that was asymptomatic (defined by radiographic findings
only) and reversible (without any anti-inflammatory therapies) is
permitted; (e) Positive test for HIV infection; (0 Active hepatitis
B (defined as having a positive hepatitis B surface antigen [HBsAg]
test at screening). Patients with past or resolved hepatitis B
infection (defined as having a negative HBsAg test and a positive
IgG antibody to hepatitis B core antigen [anti HBc]) are eligible;
(g) Active hepatitis C (patients positive for hepatitis C virus
(HCV) antibody are eligible only if PCR is negative for HCV RNA);
(h) Active tuberculosis; (i) Severe infections, including but not
limited to bacteremia, severe pneumonia, or hospitalizations for
complications of infrection within 4 weeks prior to Cycle 1, Day 1;
(j) Patients with recent infections that are not judged to be
severe are excluded if they have either: [0718] (i) Signs or
symptoms of infection within 2 weeks prior to Cycle 1, Day 1.
Patients with uncomplicated viral upper respiratory tract
infections are eligible provided symptoms have resolved to
baseline; [0719] (ii) Received oral or IV antibiotics (including
anti-fungal or anti-viral therapy) within 2 weeks prior to Cycle 1,
Day 1. Patients receiving prophylactic antibiotics (e.g., for
prevention of a urinary tract infection or chronic obstructive
pulmonary disease) are eligible; (k) Prior allogeneic bone marrow
transplantation or prior solid organ transplantation; (l)
Administration of a live, attenuated vaccine within 4 weeks before
Cycle 1, Day 1 or anticipation that such a live attenuated vaccine
may be required during the study. Influenza vaccination is given
during influenza season only. Patients must not receive live,
attenuated influenza vaccine (e.g., FluMist.RTM.) within 4 weeks
prior to Cycle 1, Day 1 or at any time during the study; and (m)
History of severe allergic, anaphylactic, or other hypersensitivity
reactions to chimeric or humanized antibodies or fusion
proteins.
[0720] Exclusion criteria specific to patients assigned to the
triplet arm include:
(a) Inadequately controlled hypertension (defined as systolic blood
pressure >150 mmHg and/or diastolic blood pressure >100 mmHg
Anti-hypertensive therapy to maintain a systolic blood pressure
<150 mmHg and/or diastolic blood pressure <100 mmHg is
permitted; (b) Prior history of hypertensive crisis or hypertensive
encephalopathy; (c) Clinically significant cardiovascular disease,
such as cerebrovascular accidents within 6 months prior to
initiation of study treatment, myocardial infarction within 6
months prior to initiation of study treatment, unstable angina, New
York Heart Association (NYHA) Grade II or greater CHF, or serious
cardiac arrhythmia uncontrolled by medication or potentially
interfering with study treatment; (d) History of stroke or
transient ischemic attack within 6 months prior to Cycle 1 Day 1;
(e) Significant vascular disease (e.g., aortic aneurysm requiring
surgical repair or recent peripheral arterial thrombosis) within 6
months prior to Cycle 1 Day 1; (f) History of Grade 4 venous
thromboembolism; (g) History of Grade 2 hemoptysis (defined as 2.5
mL of bright red blood per episode) within 3 months prior to
screening for NSCLC patients and 1 month prior to screening for
other tumor type other than NSCLC; (h) Evidence of bleeding
diathesis or clinically significant coagulopathy (in the absence of
therapeutic anticoagulation); (i) Current or recent (within 10
calendar days prior to Cycle 1 Day 1) use of dipyramidole (>400
mg/day), ticlopidine (>500 mg/day), clopidogrel (>75 mg/day),
aspirin (>325 mg/day), or cilostazol (>200 mg/day); (j)
Prophylactic or therapeutic use of low molecular weight heparin
(e.g., enoxaparin), direct thrombin inhibitors, or warfarin are
permitted, provided, where appropriate anticoagulation indices are
stable. Patients should have been on a stable dose (for therapeutic
use) for at least 2 weeks (or until reaching steady state level of
the drug) prior to the first study treatment; (k) Core biopsy or
other minor surgical procedure, excluding placement of a vascular
access device, within 7 calendar days prior to the first dose of
bevacizumab; (l) History of abdominal or tracheoesophageal fistula
or GI perforation within 6 months prior to Cycle 1 Day 1; (m)
Clinical signs or symptoms of GI obstruction or requirement for
routine parenteral hydration, parenteral nutrition, or tube
feeding; (n) Evidence of abdominal free air not explained by
paracentesis or recent surgical procedure; (o) Serious, non-healing
or dehiscing wound, active ulcer, or untreated bone fracture; (p)
Proteinuria, as demonstrated by urine dipstick or >1.0 g of
protein in a 24 hour urine collection. All patients with 2+ protein
on dipstick urinalysis at baseline must undergo a 24 hour urine
collection for protein; (q) Known hypersensitivity to any component
of bevacizumab; and (r) Intrathoracic lung cancer of squamous cell
histology. Mixed tumors are categorized by the predominant cell
type.
Dose Escalation Stage
[0721] Approximately 6 to 12 patients are enrolled in the
MOXR0916+atezolizumab+bevacizumab dose escalation study. The
starting dose of MOXR0916 is 300 mg, administered by IV infusion
every 21 days to patients in the first cohort. If the starting dose
is tolerated by the first cohort, a second cohort of 3 patients is
dosed. If the starting dose is tolerated by both cohorts (e.g., the
MTD is not exceeded), the study progresses to Expansion Part IV
(see FIG. 4B). Depending on new nonclinical efficacy, clinical
safety, and PK data, intermediate dose levels of MOXR0916 may be
evaluated. Atezolizumab is administered at a fixed dose of 1200 mg
IV every 21 days. Bevacizumab are administered at a weight-based
dose of 15 mg/kg IV every 21 days.
[0722] If the starting dose is not tolerated, or if warranted by
clinical safety or PK data, intermediate or lower dose levels of
MOXR0916 may be evaluated. Cohorts of at least 3 patients each are
treated at escalating doses of MOXR0916 in combination with a fixed
dose of atezolizumab (1200 mg) and a weight-based dose (15 mg/kg)
of bevacizumab in accordance with the dose escalation rules to
determine the MTD or maximum administered dose (MAD). Initially,
the DLT assessment window is 21 days (Days 1-21 of Cycle 1). If a
delayed DLT is observed, the DLT assessment window is extended to
42 days after the first administration of MOXR0916 and atezolizumab
for all patients in that cohort and any subsequent dose escalation
cohorts. Adverse events identified as DLTs or delayed DLTs, are
reported to the Sponsor within 24 hours.
[0723] Intrapatient dose escalation of MOXR0916 to a dose level
that has already met criteria for further escalation are allowed if
all of the following conditions are met: the patient has completed
at least 4 cycles at their originally assigned dose level and has a
documented anti therapeutic antibody titer; the patient has not
experienced a DLT or an adverse event occurring outside the DLT
window that would otherwise meet the definition of a DLT; the
patient is clinically stable with no decrement in performance
status; the Medical Monitor has approved the dose escalation.
Expansion Stage
[0724] Approximately 50-100 patients are enrolled in the expansion
stage (Part IV) to better characterize the safety, tolerability, PK
variability, biomarkers of anti-tumor activity, and preliminary
efficacy of MOXR0916 in combination with atezolizumab and
bevacizumab. The expansion stage enrolls: 20-40 patients with
treatment-naive RCC; 10-20 patients with RCC with one or more prior
lines of systemic therapy; and up to 40 patients in an additional
"basket" of patients with tumor types other than RCC that may be
opened if activity in RCC is judged to be promising (FIGS. 4A &
4B). Enrollment may be restricted on the basis of prospectively
determined tumor PD-L1 or OX40 status based on evolving biomarker
data. In this circumstance, patients whose tumor tissue is
determined to be unevaluable for expression of the pertinent
biomarker is ineligible.
Dosage, Administration, and Compliance
[0725] Administration of MOXR0916, atezolizumab, and bevacizumab is
performed in a setting with emergency medical facilities with
access to a critical care unit and staff who are trained to monitor
for and respond to medical emergencies. On days of scheduled study
treatment infusion, the order of administration is MOXR0916 (first
infusion), atezolizumab (second infusion) and bevacizumab (last
infusion), with an intervening observation period (minimum of 30
minutes).
[0726] The approximate dose levels of MOXR0916 proposed to be
evaluated in this study include 0.8, 3.2, 12, 40, 130, 300, 400,
and 1200 mg administered every 3 weeks by IV infusion. Intermediate
dose levels of MOXR0916 may be evaluated based on new nonclinical
efficacy, clinical safety, and clinical PK data after consultation
with participating investigators. Doses are not dependent on body
weight.
[0727] The initial dose of MOXR0916 is delivered over 60.+-.10
minutes (although the infusion may be slowed or interrupted for
patients who experience infusion-associated symptoms), followed by
a 30-minute observation period. If the 60-minute infusion is
tolerated without infusion-associated adverse events, all
subsequent infusions may be delivered over 30.+-.10 minutes,
followed by a 30-minute observation period. Patients who have
previously received MOXR0916 may receive the initial dose at the
fastest rate that was previously tolerated.
[0728] The dose of atezolizumab to be administered in combination
with MOXR0916 in this study is 1200 mg IV every 3 weeks. This dose
is fixed and not dependent on body weight. Atezolizumab is
administered after the MOXR0916 infusion and subsequent observation
period.
[0729] The initial dose of atezolizumab is delivered over 60.+-.10
minutes. If the first infusion is tolerated without
infusion-associated adverse events, the second infusion may be
delivered over 30.+-.10 minutes. If the 30-minute infusion is well
tolerated, all subsequent infusions may be delivered over 30
(.+-.10) minutes. All doses of atezolizumab are followed by a
30-minute observation period. Patients who have previously received
atezolizumab on another clinical trial may receive the initial dose
at the fastest rate that was previously tolerated. There is no dose
reduction for atezolizumab in this study.
[0730] The dose of bevacizumab in this study is 15 mg/kg
administered by IV infusion every 3 weeks. Body weight at baseline
is used to calculated the required dose of bevacizumab. If a weight
change of >10% from baseline is observed, the treatment dosage
is modified accordingly (i.e., this becomes the new weight for dose
calculation). Otherwise, it is not necessary to recalculate the ose
for each administration. On the day of infusion, MOXR0916 is
administered first, followed by atezolizumab and bevacizumab
infusions.
[0731] The initial dose of bevacizumab is delivered over 90.+-.10
minutes, followed by a 30-minute observation period. If the
90-minute infusion is tolerated without infusion-associated adverse
events, the second infusion may be delivered over 60.+-.10 minutes,
followed by a 30 minute observation period. If the 60-minute
infusion is well tolerated, all subsequent infusions may be
delivered over 30.+-.10 minutes, followed by a 30-minute
observation period. Bevacizumab administration may be subject to an
investigational site's standard procedures. Patients who have
previously received bevacizumab may receive the initial dose at the
fastest rate that was previously tolerated. There is no dose
reduction for bevacizumab in this study.
Outcome Measures
[0732] The safety and tolerability of MOXR0916, atezolizumab, and
bevacizumab is assessed using the following primary safety outcome
measures: incidence and nature of DLTs; and incidence, nature, and
severity of adverse events graded according to NCI CTCAE v4.0.
[0733] In addition, safety is assessed using the following
secondary safety outcome measures: incidence of anti-MOXR0916,
anti-atezolizumab, and anti-bevacizumab antibodies and the
potential correlation with PK, pharmacodynamic, and safety
parameters; change in vital signs; change in clinical laboratory
results, including ECGs; and number of cycles received and dose
intensity.
[0734] The following pharmacokinetic parameters are derived from
serum concentration-time profiles of MOXR0916 following
administration, when appropriate as data allow: AUC; Cmax; Cmin;
CL; and Vss. The following pharmacokinetic parameters are derived
from serum concentration-time profiles of atezolizumab and
bevacizumab following administration, when appropriate as data
allow: Cmax; and Cmin Other parameters such as accumulation ratio,
half-life, and dose proportionality may also be calculated.
[0735] The following activity outcome measures may be assessed:
(a) Objective response, defined as a complete response (CR) or
partial response (PR) confirmed .gtoreq.4 weeks after initial
documentation, determined using RECIST v.1.1; (b) Duration of
objective response, defined as the time from the first occurrence
of a documented, objective response until the time of relapse or
death from any cause, determined using RECIST v.1.1; (c)
Progression-free survival (PFS), defined as the time from the first
study treatment (Day 1) to the first occurrence of progression or
death from any cause, whichever occurs first, determined using
RECIST v.1.1; (d) Objective response, duration of objective
response, and PFS determined using modified RECIST; and (e) Overall
survival (OS), defined as the time from first study treatment to
death from any cause.
[0736] The following exploratory PD outcome measures may be
assessed: changes in T, B, and NK cell numbers (T, B, and NK assay)
in blood; changes in prevalence of various immune cell
subpopulations in blood (e.g., effector/memory T cells, regulatory
T cells, and MDSCs); changes in activation, proliferation, and
functional status of T-cell subsets in blood; identification and
profiling of exploratory biomarkers in plasma (i.e., interleukin-2
[IL2], IFN.gamma., and other markers); changes in
tumor-infiltrating CD8+ T cells (and other exploratory markers) in
freshly obtained tumor tissue prior to and during study treatment;
and changes in tumor-infiltrating T-cell activity (measured by
expression of granzyme B and other markers) in freshly obtained
tumor tissue prior to and during study treatment.
[0737] The following additional exploratory biomarker outcome
measures may be assessed when appropriate: status of PD-L1 and OX40
(and other exploratory markers) in tumor tissue; status of immune
infiltrate in tumor tissue, including enumeration and
characterization of various immune cell subpopulations; and
analysis of single nucleotide polymorphisms (SNPs) in genes
including but not limited to those that encode Fc receptors.
Example 4
Tumor Immune Modulation Observed in First-in-Human Phase I Dose
Escalation Study of the OX40 Agonist MOXR0916 in Patients with
Refractory Solid Tumors
[0738] In an RCC tumor biopsy from one patient that received
MOXR0916 at a dose of 3.2 mg administered in the parallel phase I
single-agent MOXR0916 study cited in reference to FIG. 2 above,
tumor immune modulation was observed. FIG. 5 shows the postdose
fold change in gene expression (as compared to predose levels) of
various immune-related genes. Upregulated genes included CCR5,
CD274, IL-7, TNFRSF14, TGFB1, CD40, CD4, PRF1, TNFSF4, CD86, CXCL9,
CD3E, LAG3, PDCD1, CCL28, GZMB, IFNg, and IL-2RA. This gene
expression pattern indicates an increase in Teff activation.
Downregulated genes included CCL22, IL-2, RORC, IL-8, CTLA4, and
FOXP3 Importantly, expression of these genes is thought to be
associated with Treg cells, thus suggesting a decrease in Treg
activity. PD-L1 expression was also assayed in the tumor biopsy
using IHC, which demonstrated an increase in PD-L1-positive area
(relative to overall tumor area) from a predose score of <1% to
a postdose score of 5%. Treg cells were also enumerated in the
tumor biopsy using immunofluorescence staining against CD3 and
Foxp3 as markers. These data indicated a predose Treg frequency
(i.e., CD3+FOXP3+ cells) of 2.15% of all cells, as compared to a
postdose frequency of 0.58%. In summary, these data indicate a
reduction in Tregs, an increase in Teff activation, and an increase
in PD-L1 expression upon MOXR0916 treatment.
[0739] In summary, these data, particularly the increase in PD-L1
expression seen after OX40 agonist treatment, suggest that MOXR0916
treatment may enhance the efficacy of, or otherwise act
synergistically with, atezolizumab treatment, e.g., through
reduction in Tregs, an increase in Teff activation, and an increase
in PD-L1 expression. The safety and complementary mechanism of
action of MOXR0916 supports its use in combination with
atezolizumab.
[0740] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, the descriptions and examples should not be
construed as limiting the scope of the invention. The disclosures
of all patent and scientific literature cited herein are expressly
incorporated in their entirety by reference.
Sequence CWU 1
1
2211249PRTHomo sapiens 1Leu His Cys Val Gly Asp Thr Tyr Pro Ser Asn
Asp Arg Cys Cys His1 5 10 15 Glu Cys Arg Pro Gly Asn Gly Met Val
Ser Arg Cys Ser Arg Ser Gln 20 25 30 Asn Thr Val Cys Arg Pro Cys
Gly Pro Gly Phe Tyr Asn Asp Val Val 35 40 45 Ser Ser Lys Pro Cys
Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly 50 55 60 Ser Glu Arg
Lys Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg65 70 75 80 Cys
Arg Ala Gly Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp 85 90
95 Cys Ala Pro Cys Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala
100 105 110 Cys Lys Pro Trp Thr Asn Cys Thr Leu Ala Gly Lys His Thr
Leu Gln 115 120 125 Pro Ala Ser Asn Ser Ser Asp Ala Ile Cys Glu Asp
Arg Asp Pro Pro 130 135 140 Ala Thr Gln Pro Gln Glu Thr Gln Gly Pro
Pro Ala Arg Pro Ile Thr145 150 155 160 Val Gln Pro Thr Glu Ala Trp
Pro Arg Thr Ser Gln Gly Pro Ser Thr 165 170 175 Arg Pro Val Glu Val
Pro Gly Gly Arg Ala Val Ala Ala Ile Leu Gly 180 185 190 Leu Gly Leu
Val Leu Gly Leu Leu Gly Pro Leu Ala Ile Leu Leu Ala 195 200 205 Leu
Tyr Leu Leu Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys 210 215
220 Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln
Ala225 230 235 240 Asp Ala His Ser Thr Leu Ala Lys Ile 245
25PRTArtificial SequenceSynthetic Construct 2Asp Ser Tyr Met Ser1 5
317PRTArtificial SequenceSynthetic Construct 3Asp Met Tyr Pro Asp
Asn Gly Asp Ser Ser Tyr Asn Gln Lys Phe Arg1 5 10 15
Glu48PRTArtificial SequenceSynthetic Construct 4Ala Pro Arg Trp Tyr
Phe Ser Val1 5 511PRTArtificial SequenceSynthetic Construct 5Arg
Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn1 5 10 67PRTArtificial
SequenceSynthetic Construct 6Tyr Thr Ser Arg Leu Arg Ser1 5
79PRTArtificial SequenceSynthetic Construct 7Gln Gln Gly His Thr
Leu Pro Pro Thr1 5 85PRTArtificial SequenceSynthetic Construct 8Asp
Ala Tyr Met Ser1 5 95PRTArtificial SequenceSynthetic Construct 9Glu
Ser Tyr Met Ser1 5 1017PRTArtificial SequenceSynthetic Construct
10Asp Met Tyr Pro Asp Asn Ala Asp Ser Ser Tyr Asn Gln Lys Phe Arg1
5 10 15 Glu1117PRTArtificial SequenceSynthetic Construct 11Asp Met
Tyr Pro Asp Asn Ala Asp Ala Ser Tyr Asn Gln Lys Phe Arg1 5 10 15
Glu1217PRTArtificial SequenceSynthetic Construct 12Asp Met Tyr Pro
Asp Asn Gly Asp Ala Ser Tyr Asn Gln Lys Phe Arg1 5 10 15
Glu1317PRTArtificial SequenceSynthetic Construct 13Asp Met Tyr Pro
Asp Ser Gly Asp Ser Ser Tyr Asn Gln Lys Phe Arg1 5 10 15
Glu1417PRTArtificial SequenceSynthetic Construct 14Asp Met Tyr Pro
Asp Asn Gly Ser Ser Ser Tyr Asn Gln Lys Phe Arg1 5 10 15
Glu158PRTArtificial SequenceSynthetic Construct 15Ala Pro Arg Trp
Tyr Phe Ser Ala1 5 168PRTArtificial SequenceSynthetic Construct
16Ala Pro Arg Trp Tyr Ala Ser Val1 5 178PRTArtificial
SequenceSynthetic Construct 17Ala Pro Arg Trp Ala Phe Ser Val1 5
188PRTArtificial SequenceSynthetic Construct 18Ala Pro Ala Trp Tyr
Phe Ser Val1 5 198PRTArtificial SequenceSynthetic Construct 19Ala
Pro Arg Trp Tyr Phe Ala Val1 5 208PRTArtificial SequenceSynthetic
Construct 20Ala Pro Arg Ala Tyr Phe Ser Val1 5 218PRTArtificial
SequenceSynthetic Construct 21Ala Ala Arg Trp Tyr Phe Ser Val1 5
229PRTArtificial SequenceSynthetic Construct 22Gln Gln Gly His Thr
Leu Pro Ala Thr1 5 239PRTArtificial SequenceSynthetic Construct
23Gln Gln Gly His Thr Ala Pro Pro Thr1 5 249PRTArtificial
SequenceSynthetic Construct 24Gln Gln Gly Ala Thr Leu Pro Pro Thr1
5 259PRTArtificial SequenceSynthetic Construct 25Gln Gln Gly His
Ala Leu Pro Pro Thr1 5 269PRTArtificial SequenceSynthetic Construct
26Gln Gln Ala His Thr Leu Pro Pro Thr1 5 279PRTArtificial
SequenceSynthetic Construct 27Gln Gln Gly His Thr Leu Ala Pro Thr1
5 289PRTArtificial SequenceSynthetic Construct 28Gln Ala Gly His
Thr Leu Pro Pro Thr1 5 295PRTArtificial SequenceSynthetic Construct
29Asn Tyr Leu Ile Glu1 5 3017PRTArtificial SequenceSynthetic
Construct 30Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe Lys1 5 10 15 Gly3117PRTArtificial SequenceSynthetic Construct
31Val Ile Asn Pro Gly Ser Gly Asp Ala Tyr Tyr Ser Glu Lys Phe Lys1
5 10 15 Gly3217PRTArtificial SequenceSynthetic Construct 32Val Ile
Asn Pro Gly Ser Gly Asp Gln Tyr Tyr Ser Glu Lys Phe Lys1 5 10 15
Gly335PRTArtificial SequenceSynthetic Construct 33Asp Arg Leu Asp
Tyr1 5 345PRTArtificial SequenceSynthetic Construct 34Ala Arg Leu
Asp Tyr1 5 355PRTArtificial SequenceSynthetic Construct 35Asp Ala
Leu Asp Tyr1 5 365PRTArtificial SequenceSynthetic Construct 36Asp
Arg Ala Asp Tyr1 5 3711PRTArtificial SequenceSynthetic Construct
37His Ala Ser Gln Asp Ile Ser Ser Tyr Ile Val1 5 10
387PRTArtificial SequenceSynthetic Construct 38His Gly Thr Asn Leu
Glu Asp1 5 397PRTArtificial SequenceSynthetic Construct 39His Gly
Thr Asn Leu Glu Ser1 5 407PRTArtificial SequenceSynthetic Construct
40His Gly Thr Asn Leu Glu Glu1 5 417PRTArtificial SequenceSynthetic
Construct 41His Gly Thr Asn Leu Glu Gln1 5 429PRTArtificial
SequenceSynthetic Construct 42Val His Tyr Ala Gln Phe Pro Tyr Thr1
5 439PRTArtificial SequenceSynthetic Construct 43Ala His Tyr Ala
Gln Phe Pro Tyr Thr1 5 449PRTArtificial SequenceSynthetic Construct
44Val Ala Tyr Ala Gln Phe Pro Tyr Thr1 5 459PRTArtificial
SequenceSynthetic Construct 45Val His Ala Ala Gln Phe Pro Tyr Thr1
5 469PRTArtificial SequenceSynthetic Construct 46Val His Tyr Ala
Ala Phe Pro Tyr Thr1 5 479PRTArtificial SequenceSynthetic Construct
47Val His Tyr Ala Gln Ala Pro Tyr Thr1 5 489PRTArtificial
SequenceSynthetic Construct 48Val His Tyr Ala Gln Phe Ala Tyr Thr1
5 499PRTArtificial SequenceSynthetic Construct 49Val His Tyr Ala
Gln Phe Pro Ala Thr1 5 505PRTArtificial SequenceSynthetic Construct
50Asp Tyr Gly Val Leu1 5 5116PRTArtificial SequenceSynthetic
Construct 51Met Ile Trp Ser Gly Gly Thr Thr Asp Tyr Asn Ala Ala Phe
Ile Ser1 5 10 15 525PRTArtificial SequenceSynthetic Construct 52Glu
Glu Met Asp Tyr1 5 5311PRTArtificial SequenceSynthetic Construct
53Arg Ala Ser Gln Asp Ile Ser Asn Phe Leu Asn1 5 10
547PRTArtificial SequenceSynthetic Construct 54Tyr Thr Ser Arg Leu
His Ser1 5 559PRTArtificial SequenceSynthetic Construct 55Gln Gln
Gly Asn Thr Leu Pro Trp Thr1 5 56117PRTArtificial SequenceSynthetic
Construct 56Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Gly
Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile
Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu
Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser 115 57107PRTArtificial SequenceSynthetic
Construct 57Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp
Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro 85 90 95 Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 58117PRTArtificial
SequenceSynthetic Construct 58Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met
Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg
Glu Arg Val Thr Ile Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75
80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Val Leu Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly
Thr Leu 100 105 110 Val Thr Val Ser Ser 115 59107PRTArtificial
SequenceSynthetic Construct 59Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr
Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
60117PRTArtificial SequenceSynthetic Construct 60Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Asp Met Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn Gln Lys
Phe 50 55 60 Arg Glu Arg Val Thr Leu Thr Val Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Pro Arg Trp Tyr Phe Ser
Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
61107PRTArtificial SequenceSynthetic Construct 61Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly
His Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105 62117PRTArtificial SequenceSynthetic Construct
62Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Ser 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Gly Asp Ser Ser
Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile Thr Val Asp
Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Pro Arg
Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val
Ser Ser 115 63107PRTArtificial SequenceSynthetic Construct 63Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Thr Val Lys Leu
Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Gly His Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys 100 105 64117PRTArtificial SequenceSynthetic
Construct 64Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Gly
Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile
Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu
Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser 115 65107PRTArtificial SequenceSynthetic
Construct 65Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp
Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro 85 90 95 Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 66117PRTArtificial
SequenceSynthetic Construct 66Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20
25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn
Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile Thr Val Asp Thr Ser
Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Pro Arg Trp Tyr
Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser
115 67107PRTArtificial SequenceSynthetic Construct 67Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25
30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Thr Val Lys Leu Leu Ile
35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln
Gly His Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys 100 105 68117PRTArtificial SequenceSynthetic Construct
68Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Ser 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Gly Asp Ser Ser
Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile Thr Val Asp
Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Pro Arg
Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val
Ser Ser 115 69107PRTArtificial SequenceSynthetic Construct 69Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Thr Val Lys Leu
Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys
Gln Gln Gly His Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys 100 105 70117PRTArtificial SequenceSynthetic
Construct 70Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Asp Ala 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Gly
Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile
Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu
Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser 115 71107PRTArtificial SequenceSynthetic
Construct 71Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp
Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro 85 90 95 Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 72117PRTArtificial
SequenceSynthetic Construct 72Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Glu Ser 20 25 30 Tyr Met Ser Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met
Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg
Glu Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75
80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Val Leu Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly
Thr Leu 100 105 110 Val Thr Val Ser Ser 115 73107PRTArtificial
SequenceSynthetic Construct 73Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr
Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
74117PRTArtificial SequenceSynthetic Construct 74Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Asp Met Tyr Pro Asp Asn Ala Asp Ser Ser Tyr Asn Gln Lys
Phe 50 55 60 Arg Glu Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Pro Arg Trp Tyr Phe Ser
Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
75107PRTArtificial SequenceSynthetic Construct 75Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly
His Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105 76117PRTArtificial SequenceSynthetic Construct
76Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Ser 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Ala Asp Ala Ser
Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile Thr Arg Asp
Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Pro Arg
Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val
Ser Ser 115 77107PRTArtificial SequenceSynthetic Construct 77Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Gly His Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys 100 105 78117PRTArtificial SequenceSynthetic
Construct 78Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Gly
Asp Ala Ser Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile
Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu
Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser 115 79107PRTArtificial SequenceSynthetic
Construct 79Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp
Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro 85 90 95 Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 80117PRTArtificial
SequenceSynthetic Construct 80Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met
Tyr Pro Asp Ser Gly Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg
Glu Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75
80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Val Leu Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly
Thr Leu 100 105 110 Val Thr Val Ser Ser 115 81107PRTArtificial
SequenceSynthetic Construct 81Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr
Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
82117PRTArtificial SequenceSynthetic Construct 82Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Asp Met Tyr Pro Asp Asn Gly Ser Ser Ser Tyr Asn Gln Lys
Phe 50 55 60 Arg Glu Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Pro Arg Trp Tyr Phe Ser
Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
83107PRTArtificial SequenceSynthetic Construct 83Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly
His Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105 84117PRTArtificial SequenceSynthetic Construct
84Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Ala 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Ala Asp Ala Ser
Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile Thr Arg Asp
Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Pro Arg
Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val
Ser Ser 115 85107PRTArtificial SequenceSynthetic Construct 85Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Gly His Thr
Leu Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 86117PRTArtificial SequenceSynthetic Construct 86Glu Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20
25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn
Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile Thr Arg Asp Thr Ser
Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Pro Arg Trp Tyr
Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser
115 87107PRTArtificial SequenceSynthetic Construct 87Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25
30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Gly His Thr Leu Pro Ala 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys 100 105 88117PRTArtificial SequenceSynthetic Construct
88Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Ser 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Gly Asp Ser Ser
Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile Thr Arg Asp
Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Pro Arg
Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val
Ser Ser 115 89107PRTArtificial SequenceSynthetic Construct 89Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Gly His Thr Ala Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys 100 105 90117PRTArtificial SequenceSynthetic
Construct 90Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Gly
Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile
Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu
Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser 115 91107PRTArtificial SequenceSynthetic
Construct 91Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp
Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Gly Ala Thr Leu Pro Pro 85 90 95 Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 92117PRTArtificial
SequenceSynthetic Construct 92Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met
Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg
Glu Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75
80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Val Leu Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly
Thr Leu 100 105 110 Val Thr Val Ser Ser 115 93107PRTArtificial
SequenceSynthetic Construct 93Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr
Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly His Ala Leu Pro Pro
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
94117PRTArtificial SequenceSynthetic Construct 94Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Asp Met Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn Gln Lys
Phe 50 55 60 Arg Glu Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Pro Arg Trp Tyr Phe Ser
Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
95107PRTArtificial SequenceSynthetic Construct 95Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala
His Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105 96117PRTArtificial SequenceSynthetic Construct
96Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Ser 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Gly Asp Ser Ser
Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile Thr Arg Asp
Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Pro Arg
Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val
Ser Ser 115 97107PRTArtificial SequenceSynthetic Construct 97Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Gly His Thr Leu Ala Pro 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys 100 105 98117PRTArtificial SequenceSynthetic
Construct 98Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Gly
Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile
Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu
Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser 115 99107PRTArtificial SequenceSynthetic
Construct 99Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp
Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Ala Gly His Thr Leu Pro Pro 85 90 95 Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 100117PRTArtificial
SequenceSynthetic Construct 100Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met
Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg
Glu Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75
80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Val Leu Ala Pro Arg Trp Tyr Phe Ser Ala Trp Gly Gln Gly
Thr Leu 100 105 110 Val Thr Val Ser Ser 115 101107PRTArtificial
SequenceSynthetic Construct 101Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr
Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
102117PRTArtificial SequenceSynthetic Construct 102Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Asp Met Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn Gln Lys
Phe 50 55 60 Arg Glu Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Pro Arg Trp Tyr Ala Ser
Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
103107PRTArtificial SequenceSynthetic Construct 103Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly
His Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105 104117PRTArtificial SequenceSynthetic Construct
104Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Ser 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Gly Asp Ser Ser
Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile Thr Arg Asp
Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Pro Arg
Trp Ala Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val
Ser Ser 115 105107PRTArtificial SequenceSynthetic Construct 105Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20
25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Gly His Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys 100 105 106117PRTArtificial SequenceSynthetic
Construct 106Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn
Gly Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr
Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val
Leu Ala Pro Ala Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105
110 Val Thr Val Ser Ser 115 107107PRTArtificial SequenceSynthetic
Construct 107Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro 85 90 95 Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 108117PRTArtificial
SequenceSynthetic Construct 108Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met
Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg
Glu Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75
80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Val Leu Ala Pro Arg Trp Tyr Phe Ala Val Trp Gly Gln Gly
Thr Leu 100 105 110 Val Thr Val Ser Ser 115 109107PRTArtificial
SequenceSynthetic Construct 109Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr
Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
110117PRTArtificial SequenceSynthetic Construct 110Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Asp Met Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn Gln Lys
Phe 50 55 60 Arg Glu Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Pro Arg Ala Tyr Phe Ser
Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
111107PRTArtificial SequenceSynthetic Construct 111Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly
His Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105 112117PRTArtificial SequenceSynthetic Construct
112Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Ser 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Gly Asp Ser Ser
Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile Thr Arg Asp
Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Ala Arg
Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val
Ser Ser 115 113107PRTArtificial SequenceSynthetic Construct 113Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Gly His Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys 100 105 114117PRTArtificial SequenceSynthetic
Construct 114Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn
Gly Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr
Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Leu Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105
110 Val Thr Val Ser Ser 115 115107PRTArtificial SequenceSynthetic
Construct 115Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro 85 90 95 Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 116117PRTArtificial
SequenceSynthetic Construct 116Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met
Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg
Glu Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75
80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Val Ala Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly
Thr Leu 100 105 110 Val Thr Val Ser Ser 115 117107PRTArtificial
SequenceSynthetic Construct 117Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr
Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
118114PRTArtificial SequenceSynthetic Construct 118Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser119107PRTArtificial
SequenceSynthetic Construct 119Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
120114PRTArtificial SequenceSynthetic Construct 120Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser121107PRTArtificial
SequenceSynthetic Construct 121Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
122114PRTArtificial SequenceSynthetic Construct 122Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser123107PRTArtificial
SequenceSynthetic Construct 123Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
124114PRTArtificial SequenceSynthetic Construct 124Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser125107PRTArtificial
SequenceSynthetic Construct 125Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
126114PRTArtificial SequenceSynthetic Construct 126Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser127107PRTArtificial
SequenceSynthetic Construct 127Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
128114PRTArtificial SequenceSynthetic Construct 128Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser129107PRTArtificial
SequenceSynthetic Construct 129Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
130114PRTArtificial SequenceSynthetic Construct 130Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser131107PRTArtificial
SequenceSynthetic Construct 131Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
132114PRTArtificial SequenceSynthetic Construct 132Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser133107PRTArtificial
SequenceSynthetic Construct 133Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Gln Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
134114PRTArtificial SequenceSynthetic Construct 134Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser135107PRTArtificial
SequenceSynthetic Construct 135Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Ala Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
136114PRTArtificial SequenceSynthetic Construct 136Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser137107PRTArtificial
SequenceSynthetic Construct 137Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Ala Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
138114PRTArtificial SequenceSynthetic Construct 138Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser139107PRTArtificial
SequenceSynthetic Construct 139Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
140114PRTArtificial SequenceSynthetic Construct 140Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser 141107PRTArtificial
SequenceSynthetic Construct 141Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
142114PRTArtificial SequenceSynthetic Construct 142Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser 143107PRTArtificial
SequenceSynthetic Construct 143Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Phe Lys Leu Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
144114PRTArtificial SequenceSynthetic Construct 144Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60
Lys Gly Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65
70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val 100 105 110 Ser Ser 145107PRTArtificial
SequenceSynthetic Construct 145Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
146114PRTArtificial SequenceSynthetic Construct 146Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser 147107PRTArtificial
SequenceSynthetic Construct 147Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Ala His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
148114PRTArtificial SequenceSynthetic Construct 148Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser149107PRTArtificial
SequenceSynthetic Construct 149Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val Ala Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
150114PRTArtificial SequenceSynthetic Construct 150Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser151107PRTArtificial
SequenceSynthetic Construct 151Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Ala Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
152114PRTArtificial SequenceSynthetic Construct 152Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser 153107PRTArtificial
SequenceSynthetic Construct 153Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Ala Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
154114PRTArtificial SequenceSynthetic Construct 154Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser 155107PRTArtificial
SequenceSynthetic Construct 155Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Ala Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
156114PRTArtificial SequenceSynthetic Construct 156Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser 157107PRTArtificial
SequenceSynthetic Construct 157Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Ala Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
158114PRTArtificial SequenceSynthetic Construct 158Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser159107PRTArtificial
SequenceSynthetic Construct 159Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Ala
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
160114PRTArtificial SequenceSynthetic Construct 160Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser 161107PRTArtificial
SequenceSynthetic Construct 161Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
162114PRTArtificial SequenceSynthetic Construct 162Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ala Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser 163107PRTArtificial
SequenceSynthetic Construct 163Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
164114PRTArtificial SequenceSynthetic Construct 164Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Ala Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser165107PRTArtificial
SequenceSynthetic Construct 165Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
166113PRTArtificial SequenceSynthetic Construct 166Glu Val Gln Leu
Val Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15 Thr Leu
Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Tyr 20 25 30
Gly Val Leu Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35
40 45 Gly Met Ile Trp Ser Gly Gly Thr Thr Asp Tyr Asn Ala Ala Phe
Ile 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln
Phe Ser Leu65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys Val 85 90 95 Arg Glu Glu Met Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser 100 105 110 Ser 167107PRTArtificial
SequenceSynthetic Construct 167Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp Ile Ser Asn Phe 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr
Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
168113PRTArtificial SequenceSynthetic Construct 168Glu Val Gln Leu
Val Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15 Thr Leu
Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Tyr 20 25 30
Gly Val Leu Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35
40 45 Gly Met Ile Trp Ser Gly Gly Thr Thr Asp Tyr Asn Ala Ala Phe
Ile 50 55 60 Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln
Val Ser Leu65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys Val 85 90 95 Arg Glu Glu Met Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser 100 105 110 Ser 169107PRTArtificial
SequenceSynthetic Construct 169Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp Ile Ser Asn Phe 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr
Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
170113PRTArtificial SequenceSynthetic Construct 170Glu Val Gln Leu
Val Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15 Thr Leu
Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Tyr 20 25 30
Gly Val Leu Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35
40 45 Gly Met Ile Trp Ser Gly Gly Thr Thr Asp Tyr Asn Ala Ala Phe
Ile 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln
Val Ser Leu65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys Val 85 90 95 Arg Glu Glu Met Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser 100 105 110 Ser 171107PRTArtificial
SequenceSynthetic Construct 171Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp Ile Ser Asn Phe 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr
Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
1725PRTArtificial SequenceSynthetic Construct 172Xaa Xaa Tyr Met
Ser1 5 17317PRTArtificial SequenceSynthetic Construct 173Asp Met
Tyr Pro Asp Xaa Xaa Xaa Xaa Ser Tyr Asn Gln Lys Phe Arg1 5 10 15
Glu1748PRTArtificial SequenceSynthetic Construct 174Ala Pro Arg Trp
Xaa Xaa Xaa Xaa1 5 1759PRTArtificial SequenceSynthetic Construct
175Gln Xaa Xaa Xaa Xaa Xaa Xaa Xaa Thr1 5 17617PRTArtificial
SequenceSynthetic Construct 176Val Ile Asn Pro Gly Ser Gly Asp Xaa
Tyr Tyr Ser Glu Lys Phe Lys1 5 10 15 Gly1777PRTArtificial
SequenceSynthetic Construct 177His Gly Thr Asn Leu Glu Xaa1 5
1789PRTArtificial SequenceSynthetic Construct 178Xaa Xaa Tyr Ala
Gln Phe Pro Tyr Xaa1 5 179107PRTArtificial SequenceSynthetic
Construct 179Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala
Ser Leu Gly1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln
Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp
Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Lys
Asp Tyr Phe Leu Thr Ile Ser Asn Leu Glu Gln65 70 75 80 Glu Asp Val
Ala Ala Tyr Phe Cys Gln Gln Gly His Thr Leu Pro Pro 85 90 95 Thr
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 180117PRTArtificial
SequenceSynthetic Construct 180Glu Val Gln Leu Gln Gln Ser Gly Pro
Glu Leu Val Lys Pro Gly Ala1 5 10 15 Ser Val Lys Ile Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val
Lys Gln Ser His Gly Lys Thr Leu Glu Trp Ile 35 40 45 Gly Asp Met
Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg
Glu Lys Val Thr Leu Thr Val Asp Lys Ser Ser Thr Thr Ala Tyr65 70 75
80 Met Glu Phe Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95 Val Leu Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Thr Gly
Thr Thr 100 105 110 Val Thr Val Ser Ser 115 181107PRTArtificial
SequenceSynthetic Construct 181Asp Ile Leu Met Thr Gln Ser Pro Ser
Ser Met Ser Val Ser Leu Gly1 5 10 15 Asp Thr Val Ser Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Leu Gln
Gln Lys Pro Gly Lys Ser Phe Arg Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Ala Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser65 70 75
80 Glu Asp Phe Ala Asp Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105
182114PRTArtificial SequenceSynthetic Construct 182Gln Val Gln Leu
Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Lys Val Thr Leu Thr Ala Asp Lys Ser Ser Ser
Thr Ala Tyr65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser
Ala Val Tyr Phe Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Thr Leu Thr Val 100 105 110 Ser Ser 183117PRTArtificial
SequenceSynthetic Construct 183Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met
Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg
Glu Arg Val Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75
80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Val Leu Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly
Thr Leu 100 105 110 Val Thr Val Ser Ser 115 184117PRTArtificial
SequenceSynthetic Construct 184Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met
Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg
Glu Arg Val Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75
80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Val Leu Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly
Thr Leu 100 105 110 Val Thr Val Ser Ser 115 18525PRTArtificial
SequenceSynthetic Construct 185Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser 20 25 18613PRTArtificial SequenceSynthetic Construct 186Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val1 5 10
18730PRTArtificial SequenceSynthetic Construct 187Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln1 5 10 15 Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 20 25 30
18811PRTArtificial SequenceSynthetic Construct 188Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser1 5 10 18923PRTArtificial
SequenceSynthetic Construct 189Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys 20
19015PRTArtificial SequenceSynthetic Construct 190Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr1 5 10 15
19132PRTArtificial SequenceSynthetic Construct 191Gly Val Pro Ser
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr1 5 10 15 Leu Thr
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25 30
19210PRTArtificial SequenceSynthetic Construct 192Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys1 5 10 19317PRTArtificial SequenceSynthetic
Construct 193Asp Met Tyr Pro Asp Ala Ala Ala Ala Ser Tyr Asn Gln
Lys Phe Arg1 5 10 15 Glu1948PRTArtificial SequenceSynthetic
Construct 194Ala Pro Arg Trp Ala Ala Ala Ala1 5 1959PRTArtificial
SequenceSynthetic Construct 195Gln Ala Ala Ala Ala Ala Ala Ala Thr1
5 19610PRTArtificial SequenceSynthetic Construct 196Gly Phe Thr Phe
Ser Asp Ser Trp Ile His1 5 10 19718PRTArtificial SequenceSynthetic
Construct 197Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala
Asp Ser Val1 5 10 15 Lys Gly1989PRTArtificial SequenceSynthetic
Construct 198Arg His Trp Pro Gly Gly Phe Asp Tyr1 5
19911PRTArtificial SequenceSynthetic Construct 199Arg Ala Ser Gln
Asp Val Ser Thr Ala Val Ala1 5 10 2005PRTArtificial
SequenceSynthetic Construct 200Ser Ala Ser Phe Lys1 5
2019PRTArtificial SequenceSynthetic Construct 201Gln Gln Tyr Leu
Tyr His Pro Ala Thr1 5 202118PRTArtificial SequenceSynthetic
Construct 202Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Asp Ser 20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Trp Ile Ser Pro Tyr Gly
Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr
Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80 Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105
110 Leu Val Thr Val Ser Ser 115 203122PRTArtificial
SequenceSynthetic Construct 203Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30 Trp Ile His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Trp Ile
Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75
80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90
95 Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys 115 120
204106PRTArtificial SequenceSynthetic Construct 204Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Ser Ala Ser Phe Lys Gly Val Pro Ser Arg Phe Ser Gly Ser
Gly 50 55 60 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro Glu Asp65 70 75 80 Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr
His Pro Ala Thr Phe 85 90 95 Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 105 205441PRTArtificial SequenceSynthetic Construct 205Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser
20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr
Ser Lys Asn Thr Ala Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg His Trp Pro
Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145
150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu Gln 165 170 175 Ser Ser Gly Ser Leu Ser Ser Val Val Thr Val Pro
Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe225 230 235 240 Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265
270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285 Lys Pro Arg Glu Glu Gln Tyr Ala Ser Tyr Val Val Ser Val
Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val305 310 315 320 Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg 340 345 350 Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser385 390
395 400 Phe Phe Lys Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn 405 410 415 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr 420 425 430 Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
206212PRTArtificial SequenceSynthetic Construct 206Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Ser Ala Ser Phe Lys Gly Val Pro Ser Arg Phe Ser Gly Ser
Gly 50 55 60 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro Glu Asp65 70 75 80 Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr
His Pro Ala Thr Phe 85 90 95 Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg Thr Val Ala Ala Pro Ser 100 105 110 Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu Lys Ser Gly Thr Ala 115 120 125 Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val 130 135 140 Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser145 150 155 160
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr 165
170 175 Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
Cys 180 185 190 Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
Ser Phe Asn 195 200 205 Arg Gly Glu Cys 210 20732PRTArtificial
SequenceSynthetic Construct 207Arg Phe Thr Ile Ser Ala Asp Thr Ser
Lys Asn Thr Ala Tyr Leu Gln1 5 10 15 Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 20811PRTArtificial
SequenceSynthetic Construct 208Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ala1 5 10 209118PRTArtificial SequenceSynthetic Construct
209Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp
Ser 20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp
Thr Ser Lys Asn Thr Ala Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg His Trp
Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr
Val Ser Ala 115 210434PRTArtificial SequenceSynthetic Construct
210Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1
5 10 15 Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn
Ser 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Phe65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asn Asp Asp
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser 115 120 125 Arg
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135
140 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr145 150 155 160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Ser Leu 165 170 175 Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Lys Thr Tyr 180 185 190 Thr Cys Asn Val Asp His Lys Pro
Ser Asn Thr Lys Val Asp Lys Arg 195 200 205 Val Glu Ser Lys Tyr Gly
Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu 210 215 220 Phe Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp225 230 235 240 Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 245 250
255 Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
260 265 270 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe Asn 275 280 285 Ser Tyr Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn 290 295 300 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Gly Leu Pro Ser Ser305 310 315 320 Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln 325 330 335 Val Tyr Thr Leu Pro
Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 340 345 350 Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 355 360 365 Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 370 375
380 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Lys Arg Leu Thr Val
Asp385 390 395 400 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
Ser Val Met His 405 410 415 Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Leu 420 425 430 Gly Lys 211212PRTArtificial
SequenceSynthetic Construct 211Glu Ile Val Leu Thr Gln Ser Pro Ala
Thr Leu Ser Leu Ser Pro Gly1 5 10 15 Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Asn
Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly 50 55 60 Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp65 70 75
80 Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg Thr Phe
85 90 95 Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
Pro Ser 100 105 110 Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly Thr Ala 115 120 125 Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala Lys Val 130 135 140 Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln Glu Ser145 150 155 160 Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr 165 170 175 Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys 180 185 190 Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn 195 200
205 Arg Gly Glu Cys 210 212441PRTArtificial SequenceSynthetic
Construct 212Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys
Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Asn Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asn Pro Ser Asn
Gly Gly Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Asn Arg Val Thr
Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr65 70 75 80 Met Glu Leu
Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln 100 105
110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr
Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Lys
Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205 Asn Thr Lys
Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys 210 215 220 Pro
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu225 230
235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro
Glu Val Gln 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Phe Asn Ser Tyr
Val Val Ser Val Leu Thr Val 290 295 300 Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser305 310 315 320 Asn Lys Gly Leu
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys 325 330 335 Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu 340 345 350
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355
360 365 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu 370 375 380 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe385 390 395 400 Phe Lys Arg Leu Thr Val Asp Lys Ser Arg
Trp Gln Glu Gly Asn Val 405 410 415 Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln 420 425 430 Lys Ser Leu Ser Leu Ser
Leu Gly Lys 435 440 213218PRTArtificial SequenceSynthetic Construct
213Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1
5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr
Ser 20 25 30 Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro 35 40 45 Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu
Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser65 70 75 80 Ser Leu Glu Pro Glu Asp Phe
Ala Val Tyr Tyr Cys Gln His Ser Arg 85 90 95 Asp Leu Pro Leu Thr
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135
140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
Ser145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val
Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 214108PRTArtificial
SequenceSynthetic Construct 214Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45 Tyr Phe Thr
Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
215123PRTArtificial SequenceSynthetic Construct 215Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp
Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser
Thr Ala Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro His Tyr Tyr Gly Ser
Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 115 120 21610PRTArtificial SequenceSynthetic
Construct 216Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn1 5 10
21717PRTArtificial SequenceSynthetic Construct 217Trp Ile Asn Thr
Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe Lys1 5 10 15
Arg21814PRTArtificial SequenceSynthetic Construct 218Tyr Pro His
Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val1 5 10
21911PRTArtificial SequenceSynthetic Construct 219Ser Ala Ser Gln
Asp Ile Ser Asn Tyr Leu Asn1 5 10 2207PRTArtificial
SequenceSynthetic Construct 220Phe Thr Ser Ser Leu His Ser1 5
2219PRTArtificial SequenceSynthetic Construct 221Gln Gln Tyr Ser
Thr Val Pro Trp Thr1 5
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